Composition for treating ectopic calcification disorders and method for using the same

Abstract

Novel compositions and methods for treating diseases and disorders associated with pathological calcification and / or pathological ossification are provided. [Solution] A composition having the structure export-protein-Z-domain-XY, where export is absent or is a transport signal sequence or a biologically active fragment thereof, the protein is the extracellular domain of ENPP3 or a biologically active fragment thereof, the domain is selected from the group consisting of a human IgG Fc domain and a human albumin domain, X and Z are independently absent or are polypeptides comprising 1 to 20 amino acids, and Y is absent or is a polypeptide having a specific sequence, and a method of using the same.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 257,883, filed on November 20, 2015, pursuant to 35 U.S.C. 119(e). The said Provisional Application is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Calcification is the accumulation of calcium salts in body tissues. While calcification usually occurs during bone formation, calcium can also be abnormally deposited in soft tissues such as arteries, cartilage, and heart valves. Vascular calcification is common in patients with atherosclerosis, stroke, valvular heart disease, and varicose veins. Advanced age and metabolic disorders, including diabetes, are contributing factors.

[0003] Ossification is the process of bone tissue formation or bone remodeling carried out by osteoblasts. When ossification occurs, bone formation becomes possible while the fetus is still in the womb, and various types of connective tissue are converted into bone. The two main ossification processes are intramembranous ossification and endochondral ossification, which differ based on the location in the body where cartilage is present.

[0004] Abnormal levels of calcification and ossification can lead to a range of diseases. Some examples of such diseases include general arterial calcification of infancy (GACI), idiopathic infantile arterial calcification (IIAC), pseudoxanthoma elasticum (PXE), ossification of the posterior longitudinal ligament (OPLL), medial wall vascular calcification (MWVC), autosomal recessive hypophosphatemic rickets type 2 (ARHR2), end-stage renal disease (ESRD), chronic kidney disease-bone / mineral disorder (CKD-MBD), X-linked hypophosphatemia (XLH), age-related osteopenia, calcific uremic arteriolopathy (CUA), and hypophosphatemic rickets.

[0005] GACI is an extremely rare neonatal disease characterized by widespread arterial calcification in large and medium-sized vessels that develops in infancy, resulting in cardiovascular collapse and death in the neonatal period. Clinically, the disease presents with heart failure, dyspnea, hypertension, cyanosis, and cardiac hypertrophy. The prognosis is severe, with older reports indicating an 85% mortality rate at 6 months, whereas recent intensive treatment with bisphosphonates (e.g., etidronate) has reduced the 6-month mortality rate to 55%. This significant progress is hindered by severe skeletal toxicity associated with long-term use of etidronate in GACI patients, and even when initiated early, bisphosphonates were ineffective in preventing death in some patients. Furthermore, due to limited available data, it is difficult to determine whether bisphosphonate treatment truly provides protection or whether the patient response is inconsistent with the natural history of the disease. Interestingly, serum PPi levels appear to be significantly reduced in GACI patients.

[0006] The kidneys are essential for maintaining normal bone and mineral metabolism, including phosphate excretion. As revealed by the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKFK / DOQI), in 2003, 19.5 million American adults had chronic kidney disease (CKD), and 13.6 million had CKD in stages 2-5. The incidence of end-stage renal disease (ESRD) is increasing at an alarming rate. In 2000, more than 90,000 people in the United States had end-stage renal disease. The population of patients undergoing dialysis or requiring transplantation reached 380,000 in 2003 and 651,000 in 2010. In the United States, more than $18 billion is already spent annually on treating ESRD patients, which is a significant burden on the healthcare system. Importantly, patients with renal failure are unable to properly regulate the balance of minerals in their serum and tend to retain phosphate absorbed from various food components. High serum phosphate levels are associated with excessive parathyroid hormone secretion and a tendency for soft tissues, including blood vessels, to calcify.

[0007] In patients with renal failure, phosphate and pyrophosphate ions may be excessively removed during hemodialysis or peritoneal dialysis. Depletion of these anions from tissues and plasma leads to impaired bone and mineral metabolism, including osteomalacia, soft tissue calcification, and bone disease. Calcium deposition in small blood vessels in the skin causes inflammatory vasculitis called calciphylaxis, which can lead to gangrene of the skin and underlying tissues, causing severe chronic pain. Calciphylaxis may require amputation of the affected limbs, there is no effective treatment for this condition, and it is generally fatal. Therefore, it is important to regulate pyrophosphate levels in the system and suppress the development of calciphylaxis in patients.

[0008] CUA is a fatal disorder seen in CKD patients undergoing dialysis. Calcification of small arteries leads to tissue / cutaneous ischemia, infarction, and thrombosis, resulting in a mortality rate of nearly 80%. Currently, there are 450,000 dialysis patients at risk of developing CUA in the United States, and there is no FDA-approved treatment for the disorder. CUA has distinctive features similar to GACI and other calcification disorders, showing low levels of PPi and high levels of fibroblast growth factor 23 (i.e., FGF23). In ESRD patients requiring dialysis, this calcification process is more accelerated, with an average life expectancy of 5-6 years.

[0009] PXE is a genetic disorder characterized by elastic fiber calcification in the skin, arteries, and retina, leading to skin lesions accompanied by flaccidity and loss of elasticity, arterial insufficiency, cardiovascular disease, and retinal hemorrhages that can result in macular degeneration. Mutations associated with PXE are also located in the abcc6 gene. Generally, characteristic skin lesions (yellowish papules and plaques, as well as flaccidity and loss of elasticity, typically found on the face, neck, axillae, anterior cubital fossa, popliteal fossa, groin, and perimalis region) are early signs of PXE, resulting from the accumulation of abnormally calcified elastic fibers in the middermis. Skin lesions are usually detected in childhood or adolescence and progress slowly, often unpredictably. A diagnosis of PXE can be confirmed by a skin biopsy showing fragmented elastic fiber calcification in the mid and lower dermis. While skin symptoms are among the most common features of PXE, ocular and cardiovascular symptoms are responsible for the pathological state of this disease.

[0010] Common cardiovascular complications of PXE are due to the presence of abnormally calcified elastic fibers in the internal elastic lamina of medium-sized arteries. A wide range of phenotypes include premature atherosclerotic changes, intimal fibrosis causing angina or intermittent claudication, or both, early myocardial infarction, and hypertension. Restrictive cardiomyopathy may also occur due to fibrous thickening of the endocardium and atrioventricular valves. Approximately 10% of PXE patients also develop gastrointestinal bleeding and central nervous system complications (e.g., stroke and dementia) as a result of systemic arterial wall calcification. Furthermore, renovascular hypertension and atrial septal artery aneurysms can be observed in PXE patients.

[0011] Low serum phosphate levels are called hypophosphatemia, and high serum phosphate levels are called hyperphosphatemia. Hypophosphatemia is often caused by renal phosphate wasting and is triggered by numerous genetic disorders, including X-linked hypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets with hypercalciuria (HHRH), hypophosphatemic bone disease (HBD), and autosomal dominant hypophosphatemic rickets (ADHR). The precise molecular mechanisms by which appropriate serum phosphate concentrations are maintained are not fully understood.

[0012] Novel compositions and methods for treating diseases and disorders associated with pathological calcification and / or pathological ossification are needed in the art. Such compositions and methods should not unnecessarily disrupt other physiological processes. Such compositions and methods should reduce calcification levels and increase PPi plasma levels in individuals exhibiting plasma PPi levels lower than normal plasma PPi levels. The present invention satisfies this need. [Brief explanation of the drawing]

[0013] The following detailed description of exemplary embodiments of the present invention will be better understood in conjunction with the accompanying drawings. Exemplary embodiments are shown in the drawings to illustrate the present invention. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings.

[0014] [Figure 1A] Figures 1A-1C include graphs illustrating studies of the steady-state ATP hydrolysis activity of human ENPP3. Figure 1A shows the time course of AMP product formation after adding 0.98 μM, 1.95 μM, 3.9 μM, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM ATP (from bottom to top) to 50 nM hNPP3. The enzymatic reaction was stopped at various time points with equal amounts of 3 M formic acid, and the reaction product AMP was quantified by HPLC analysis using an AMP calibration curve. Flat lines passing through the data points represent the best fit with a nonlinear enzyme kinetic model with product inhibition and substrate depletion. [Figure 1B] Figure 1B illustrates a comparison of steady-state ATPase cycling rates. The dependence of the enzyme cycling rate at initial steady state on ENPP3 substrate concentration was compared to values ​​previously measured for human ENPP1. The ATPase cycling reactions of 50 nM hNPP3 and hNPP1 completely depleted the ATP substrate in 1 minute at 0.98 μM, 1.95 μM, and 3.9 μM ATP. Therefore, these three rates could not be accurately determined and were excluded from the plot. The steady-state ATPase reaction of hNPP3 reached a maximum value (kcat) of 2.59 (±0.04) s-1 enzyme-1 from the weighted average of rate measurements at substrate concentrations of 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, and 125 μM. This appears to be slower than the 3.46 (±0.44) s-1 enzyme-1 of hNPP1. The kM can be estimated to be <8 μM. With a substrate concentration of >125 μM [ATP], the ATPase turnover rate of hNPP3 gradually decreased. [Figure 1C]Figure 1C illustrates the substrate concentration-dependent η. Since the η value decreases with increasing substrate concentration for both enzymes, it can be seen that substrate depletion contributes far more to the nonlinearity in the enzyme reaction time course than product inhibition at low initial substrate concentrations. The remarkable similarity between the η values ​​of human ENPP3 and human ENPP1 indicates that the reaction rates and product inhibition of the two enzymes are similar. The rate of hNPP1 is slightly faster, and therefore, hNPP1 depletes substrate ATP slightly faster than hNPP3 at low substrate concentrations. [Figure 2] This figure illustrates the non-limiting purification profile of NPP3 fusion protein by Coomassie-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In this figure, the purified NPP3 protein is shown compared to a specific size marker. [Figure 3] A non-restrictive plasmid construct map of human NPP121-NPP3-Fc in plasmids cloned using the indicated restriction endonuclease sites is illustrated. [Figure 4] This figure illustrates a non-restrictive plasmid construct map of human NPP121-NPP3-Fc in plasmid pcDNA3, cloned using IN-FUSION® technology. [Figure 5] A non-restrictive plasmid construct map of human NPP121-NPP3-albumin in plasmid pcDNA3 is illustrated. [Overview of the Initiative]

[0015] The present invention provides an isolated polypeptide or a pharmaceutically acceptable salt or solvate thereof. Furthermore, the present invention provides a method for treating or preventing a disease or disorder associated with pathological calcification or pathological ossification in a subject in need of such treatment or prevention. Furthermore, the present invention provides a method for reducing or preventing vascular calcification in a subject with low plasma pyrophosphate (PPi) or high serum phosphate (Pi). Furthermore, the present invention provides a method for treating a subject having NPP1 deficiency or an NPP1-related disease. Furthermore, the present invention provides a kit comprising at least one isolated polypeptide of the present invention, and instructions describing the use of the at least one polypeptide for treating a disease or disorder associated with pathological calcification or pathological ossification in a subject in need of such treatment, and optionally further comprising an applicator.

[0016] In certain embodiments, the polypeptide of the present invention has Formula (I): EXPORT-PROTEIN-Z-DOMAIN-X-Y (I), wherein in (I), EXPORT is absent, or is a transport signal sequence or a biologically active fragment thereof, PROTEIN is the extracellular domain of ENPP3 (SEQ ID NO: 1) or a biologically active fragment thereof, DOMAIN is selected from the group consisting of a human IgG Fc domain and a human albumin domain, and X and Z are each independently absent, or are a polypeptide comprising 1 to 20 amino acids, Y is absent, or is a sequence selected from the group consisting of TIFF0007933352000001.tif31146, wherein n in each occurrence is independently an integer of 1 to 20.

[0017] In one particular embodiment, the nuclease domain of the protein or its variant is absent. In another embodiment, the export is absent or selected from the group consisting of SEQ ID NO: 2 to 5. In yet another embodiment, X is absent, selected from the group consisting of a polypeptide of 20 amino acids, a polypeptide of 19 amino acids, a polypeptide of 18 amino acids, a polypeptide of 17 amino acids, a polypeptide of 16 amino acids, a polypeptide of 15 amino acids, a polypeptide of 14 amino acids, a polypeptide of 13 amino acids, a polypeptide of 12 amino acids, a polypeptide of 11 amino acids, a polypeptide of 10 amino acids, a polypeptide of 9 amino acids, a polypeptide of 8 amino acids, a polypeptide of 7 amino acids, a polypeptide of 6 amino acids, a polypeptide of 5 amino acids, a polypeptide of 4 amino acids, a polypeptide of 3 amino acids, a polypeptide of 2 amino acids, and a polypeptide of 1 amino acid. In yet another embodiment, Z is selected from the group consisting of a polypeptide consisting of 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0018] In certain specific embodiments, the domain (DOMAIN) is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In other embodiments, the polypeptide is selected from the group consisting of SEQ ID NO: 19, 21, and 22. In still other embodiments, the domain (DOMAIN) is a human albumin domain. In still other embodiments, the polypeptide is selected from the group consisting of SEQ ID NO: 24, 25, and 26.

[0019] In certain specific embodiments, the polypeptide comprises the soluble region of NPP3 or a fusion protein thereof, lacks a transmembrane domain and a signal peptide, and the polypeptide reduces cellular calcification when administered to a subject suffering from a disease of calcification and ossification. In other embodiments, the polypeptide comprises the soluble region of NPP3, lacks a transmembrane domain and a signal peptide, and the polypeptide reduces cellular calcification when administered to a subject suffering from a disease of calcification and ossification.

[0020] In certain specific embodiments, the polypeptide comprises the extracellular domain of ENPP3 (SEQ ID NO: 1) or a biologically active fragment thereof. In other embodiments, the polypeptide consists essentially of SEQ ID NO: 1 or a biologically active fragment thereof. In still other embodiments, the polypeptide consists of SEQ ID NO: 1 or a biologically active fragment thereof.

[0021] In certain specific embodiments, the soluble ENPP3 fragment or fusion protein thereof comprises the extracellular domain of ENPP3 (SEQ ID NO: 1) or a biologically active fragment thereof. In other embodiments, the soluble ENPP3 fragment consists essentially of SEQ ID NO: 1 or a biologically active fragment thereof. In still other embodiments, the soluble ENPP3 fragment consists of SEQ ID NO: 1 or a biologically active fragment thereof. In still other embodiments, the soluble ENPP3 fragment or fusion protein thereof is free of a transmembrane domain and a signal peptide.

[0022] In one particular embodiment, the method comprises administering a therapeutically effective amount of at least one polypeptide of the present invention or a pharmaceutically effective salt or solvate thereof to a target. In another embodiment, the method comprises administering a therapeutically effective amount of an isolated recombinant human soluble ENPP3 fragment or a fusion protein thereof to a target.

[0023] In certain embodiments, the disease or disorder includes at least one selected from the group consisting of GACI, IIAC, PXE, OPLL, hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic plaques, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, age-related arteriosclerosis, and calciphylaxis resulting from end-stage renal disease (or bone mineral metabolism disorders associated with chronic kidney disease).

[0024] In certain embodiments, the disease or disorder includes at least one selected from the group consisting of GACI, IIAC, PXE, OPLL, MWVC, ARHR2, ESRD, CKD-MBD, XLH, age-related osteopenia, CUA, and hypophosphatemic rickets.

[0025] In one particular aspect, the disease or disorder is GACI. In another aspect, the disease or disorder is IIAC. In yet another aspect, the disease or disorder is PXE. In yet another aspect, the disease or disorder is OPLL. In yet another aspect, the disease or disorder is hypophosphatemic rickets. In yet another aspect, the disease or disorder is osteoarthritis. In yet another aspect, the disease or disorder is calcification of atherosclerotic lesions. In yet another aspect, the disease or disorder is hereditary and non-hereditary osteoarthritis. In yet another aspect, the disease or disorder is ankylosing spondylitis. In yet another aspect, the disease or disorder is age-related arteriosclerosis. In yet another aspect, the disease or disorder is calciphylaxis (or bone mineral metabolism disorder associated with chronic kidney disease) due to end-stage renal disease. In yet another aspect, the disease or disorder is age-related osteopenia. In yet another aspect, the disease or disorder is CUA. In yet another aspect, the disease or disorder is MWVC. In yet another embodiment, the disease or disorder is ARHR2. In yet another embodiment, the disease or disorder is ESRD.

[0026] In one particular embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 800 nM. In another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 1 μM. In yet another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 1.5 μM.

[0027] In one particular embodiment, at least one polypeptide is administered to the subject over a short or long period. In another embodiment, at least one polypeptide is administered to the subject locally, locally, or systemically. In yet another embodiment, the subject is a mammal. In yet another embodiment, the mammal is a human. [Invention 1001] Isolated polypeptide of formula (I) or its pharmaceutically acceptable salt or solvate: Export-protein-Z-domain-XY(I) During the ceremony, EXPORT is either absent or is a transport signal sequence or a biologically active fragment thereof. The protein (PROTEIN) is the extracellular domain (SEQ ID NO:1) of ENPP3 or its biologically active fragment. The domain is selected from the group consisting of human IgG Fc domains and human albumin domains. X and Z are, independently, polypeptides that are absent or contain 1 to 20 amino acids. Y does not exist, or This is an array selected from the group consisting of TIFF0007933352000002.tif37134, where n is an integer from 1 to 20, independently for each case. [Invention 1002] A polypeptide of the present invention 1001, which lacks a nuclease domain of a protein or its variant. [Invention 1003] A polypeptide according to the present invention 1001, wherein the export (EXPORT) is absent or selected from the group consisting of SEQ ID NO: 2 to 5. [Invention 1004] The polypeptide of the present invention 1001, wherein X and Z are independently selected from the group consisting of a polypeptide consisting of 20 amino acids, a polypeptide consisting of 19 amino acids, a polypeptide consisting of 18 amino acids, a polypeptide consisting of 17 amino acids, a polypeptide consisting of 16 amino acids, a polypeptide consisting of 15 amino acids, a polypeptide consisting of 14 amino acids, a polypeptide consisting of 13 amino acids, a polypeptide consisting of 12 amino acids, a polypeptide consisting of 11 amino acids, a polypeptide consisting of 10 amino acids, a polypeptide consisting of 9 amino acids, a polypeptide consisting of 8 amino acids, a polypeptide consisting of 7 amino acids, a polypeptide consisting of 6 amino acids, a polypeptide consisting of 5 amino acids, a polypeptide consisting of 4 amino acids, a polypeptide consisting of 3 amino acids, a polypeptide consisting of 2 amino acids, and a polypeptide consisting of 1 amino acid. [Invention 1005] The polypeptide of the present invention 1001, wherein the domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. [Invention 1006] A polypeptide of the present invention 1005, selected from the group consisting of SEQ ID NO: 19, 21, and 22. [Invention 1007] The polypeptide of the present invention 1001, wherein the domain is a human albumin domain. [Invention 1008] A polypeptide of the present invention 1007, selected from the group consisting of SEQ ID NO: 24, 25, and 26. [Invention 1009] An isolated polypeptide or fusion protein comprising a soluble region of NPP3 and lacking a transmembrane domain and a signal peptide, wherein the polypeptide reduces cellular calcification when administered to a subject suffering from a calcification and ossification disorder. [Invention 1010] The polypeptide of the present invention 1009, comprising the extracellular domain of ENPP3 (SEQ ID NO:1) or a biologically active fragment thereof. [Invention 1011] The polypeptide of the present invention 1010, essentially consisting of SEQ ID NO:1 or a biologically active fragment thereof. [Invention 1012] A method for treating or preventing a disease or disorder related to pathological calcification or pathological ossification in a subject requiring treatment or prevention of such a disease or disorder, comprising the step of administering to the subject a therapeutically effective amount of at least one isolated polypeptide of the present invention 1001. [Invention 1013] The method of the present invention 1012, wherein the disease or disorder comprises at least one selected from the group consisting of systemic arteriosclerosis of infants (GACI), idiopathic arteriosclerosis of infants (IIAC), pseudoxanthoma elasticum (PXE), OPLL, hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic lesions, pseudoxanthoma elasticum, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, age-related arteriosclerosis, and calciphylaxis resulting from end-stage renal disease (or bone mineral metabolism abnormalities associated with chronic kidney disease). [Invention 1014] The method of the present invention 1012, wherein the nuclease domain of the protein or its variant is absent. [Invention 1015] The method of the present invention 1012, wherein no export exists, or the export is selected from the group consisting of SEQ ID NO: 2 to 5. [Invention 1016] The method of the present invention 1012, wherein X and Z are independently selected from the group consisting of a polypeptide comprising 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid, and X and Z are absent. [Invention 1017] The method of the present invention 1012, wherein at least one of the polypeptides is administered to the subject over a short or long period of time. [Invention 1018] The method of the present invention 1012, wherein at least one polypeptide is administered locally, locally, or systemically to the subject. [Invention 1019] The method of the present invention 1012, wherein the domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. [Invention 1020] The method of the present invention 1019, wherein at least one polypeptide is selected from the group consisting of SEQ ID NO: 19, 21, and 22. [Invention 1021] The method of the present invention 1012, wherein the domain is a human albumin domain. [Invention 1022] The method of the present invention 1021, wherein at least one polypeptide is selected from the group consisting of SEQ ID NO: 24, 25, and 26. [Invention 1023] The method of the present invention 1012, wherein the subject is a mammal. [Invention 1024] The method of the present invention 1023, wherein the mammal is a human. [Invention 1025] A method for reducing or preventing vascular calcification in subjects with low plasma pyrophosphate (PPi) levels or high serum phosphate (Pi) levels, comprising the step of administering a therapeutically effective amount of an isolated recombinant human soluble ENPP3 fragment or fusion protein thereof to the subject, wherein the administered amount raises the plasma PPi level in the subject to at least about 800 nM. [Invention 1026] The method of the present invention 1025, wherein the amount administered increases the plasma PPi level in the subject to at least about 1 μM. [Invention 1027] The method of the present invention 1026, wherein the amount administered increases the plasma PPi level in the subject to at least about 1.5 μM. [Invention 1028] The method of the present invention 1025, wherein the subject has at least one disease selected from the group consisting of GACI, IIAC, PXE, OPLL, MWVC, ARHR2, ESRD, CKD-MBD, XLH, age-related osteopenia, CUA, and hypophosphatemic rickets. [Invention 1029] The method of the present invention 1025, wherein the soluble ENPP3 fragment or fusion protein comprises the extracellular domain of ENPP3 (SEQ ID NO:1) or a biologically active fragment thereof. [Invention 1030] The method of the present invention 1025, wherein the soluble ENPP3 fragment essentially consists of SEQ ID NO:1 or a biologically active fragment thereof. [Invention 1031] The method of the present invention 1025, wherein the soluble ENPP3 fragment or its fusion protein lacks a transmembrane domain and signaling pathway. [Invention 1032] A method for treating a subject having NPP1 deficiency or an NPP1-related disease, comprising the step of administering a therapeutically effective amount of an isolated recombinant human soluble ENPP3 fragment or fusion protein thereof to the subject. [Invention 1033] The method of the present invention 1032, wherein the subject has at least one disease selected from the group consisting of GACI, IIAC, PXE, OPLL, MWVC, ARHR2, ESRD, CKD-MBD, XLH, age-related osteopenia, CUA, and hypophosphatemic rickets. [Invention 1034] The method of the present invention 1032, wherein the soluble ENPP3 fragment or fusion protein comprises the extracellular domain of ENPP3 (SEQ ID NO:1) or a biologically active fragment thereof. [Invention 1035] The method of the present invention 1032, wherein the soluble ENPP3 fragment essentially consists of SEQ ID NO:1 or a biologically active fragment thereof. [Invention 1036] The method of the present invention 1032, wherein the soluble ENPP3 fragment or its fusion protein lacks a transmembrane domain and a signal peptide. [Invention 1037] A kit comprising at least one isolated polypeptide of any of invention 1001 to 1011, and instructions for the use of the at least one polypeptide for treating a disease or disorder related to pathological calcification or pathological ossification in subjects requiring treatment of such disease or disorder. [Invention 1038] The kit of the present invention 1037, wherein the disease or disorder comprises at least one selected from the group consisting of GACI, IIAC, OPLL, XLH, osteoarthritis, calcification of atherosclerotic lesions, pseudoxanthoma elasticum, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, age-related arteriosclerosis, calciphylaxis due to end-stage renal disease (or CKD-MBD), MWVC, ARHR2, ESRD, age-related osteopenia, and CUA. [Modes for carrying out the invention]

[0028] Detailed description of the invention This invention relates to the discovery that ENPP3 (also known as NPP3), a member of the ectonucleotide pyrophosphatase / phosphodiesterase (ENPP or NPP) enzyme family, possesses potent ATP hydrolase activity. As demonstrated herein, ENPP3 hydrolyzes ATP to AMP and PPi.

[0029] In certain aspects, the present invention provides compositions, including but not limited to fusion proteins, that increase plasma PPi in a physiological state of low plasma PPi that puts an individual at risk of pathological conditions associated with a low PPi state (as confirmed, for example, by a healthcare professional or by consulting medical documents or manuals). In certain embodiments, these physiological conditions are recognized disease conditions such as GACI, PXE, Hutchinson-Gilford Progeria Syndrome, chronic kidney disease (CKD), X-linked hypophosphatemia, sickle cell anemia, and end-stage renal disease. In other embodiments, these physiological conditions are non-disease conditions that occur in elderly individuals with chronic diseases known to occur in all elderly people, such as arteriosclerosis and osteopenia.

[0030] In certain embodiments, hypoplasmic PPi is defined as a plasma PPi concentration of less than approximately 1.5 μM. These disease conditions may or may not be associated with pathological calcification of arteries and / or soft tissues, vascular wall calcification, stroke or cerebrovascular disease, decreased pulse wave velocity, calcification of soft tissues, e.g., skin, Bruch's membrane in the eye, calcification of soft tissues around tendons, also known as tendon attachments, calcification of ligaments in the spine, e.g., posterior longitudinal ligament, and ossifying diseases, e.g., rickets. In other embodiments, the present invention includes treatment of hypoPPi physiological conditions via administration of the fusion protein described herein.

[0031] In other contexts, the compositions and methods of the present invention can be used to treat disease conditions known to occur in conditions of reduced expression or activity of the enzyme ENPP1. These recognized disease conditions include, but are not limited to, osteoarthritis, GACI, and ARHR2. These conditions may also occur in other physiological conditions of reduced ENPP1 protein levels, for example, in individuals with a common polymorphism (polymporphism) in the ENPP1 coding region, where the K residue at position 121 of the secreted protein (or position 173 of the full-length protein) is substituted with a Q residue (Eller, et al., 2008, Nephrol. Dial. Transplant. 23(1):321-7; Flanagan, et al., 2013, Blood 121(16):3237-45).

[0032] As demonstrated herein, to test the enzymatic activity of ENPP3, the products of ATP hydrolysis by this enzyme and the corresponding enzymatic constants were analyzed. ENPP3 was found to be a potent ATP hydrolase capable of producing PPi and AMP from ATP. In certain embodiments, ENPP3 has ATP hydrolase activity comparable to that of ENPP1. As demonstrated herein, ENPP3 catalyzes the hydrolysis of ATP to PPi with nearly the same Michaelis-Menten rate equation as ENPP1, another member of the ENPP enzyme family. In certain embodiments, soluble fusion constructs of ENPP3, including its albumin fusion construct and / or its IgG Fc domain construct, are effective in treating disorders of ectopic calcification. In yet other embodiments, the constructs described herein are effective in treating and / or preventing disorders of ectopic vascular calcification.

[0033] In one aspect, NPP3 is not adequately transported to the cell surface. In a particular embodiment, a soluble ENPP3 protein is constructed by exchanging the signal sequence of NPP3 with the intrinsic signal sequence of another ENPP. In another embodiment, a soluble ENPP3 construct is prepared by using the transport signal sequences of other ENPP enzymes, such as but not limited to ENPP7 and / or ENPP5. In yet another embodiment, a soluble ENPP3 construct is prepared by using a signal sequence consisting of a combination of the signal sequences of ENPP1 and ENPP2 (hereinafter, "ENPP1-2-1"). In yet another embodiment, signal sequences of any other known protein, such as but not limited to the signal sequences of immunoglobulin κ light chain protein and λ light chain protein, can also be used to target the extracellular domain of ENPP3 for secretion. Furthermore, the present invention should not be construed as being limited to the constructs described herein, but also includes constructs comprising any enzymatically active ENPP3 extracellular domain cleavage.

[0034] Diseases and disorders involving pathological calcification and / or pathological ossification that can be treated by the compositions and methods of the present invention include, but are not limited to, idiopathic infantile arterial calcification (IIAC), ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic plaques, pseudoxanthoma elasticum (PXE), hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, age-related arteriosclerosis, and calciphylaxis resulting from end-stage renal disease.

[0035] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described.

[0036] As used herein, the following terms each have their respective meanings in this section.

[0037] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “element” means one or more elements.

[0038] When used in the context of organisms, tissues, cells, or components thereof, the term “abnormal” refers to an organism, tissue, cell, or component thereof that differs from the “normal” (expected) respective characteristic in respect of at least one observable or detectable feature (e.g., age, treatment, time of day). A characteristic that is normal or expected for one cell or tissue type may be abnormal for a different cell or tissue type.

[0039] As used herein, "about" means, when referring to a measurable value, such as a quantity or period, to include a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from a specified value, if such variation is suitable for carrying out the disclosed method.

[0040] As used herein, the term "ADHR" refers to autosomal dominant hypophosphatemic rickets.

[0041] As used herein, the term “albumin” refers to a plasma protein produced in the liver that forms a large proportion of total plasma proteins. In certain embodiments, albumin refers to human serum albumin. The use of other albumins, such as bovine serum albumin, horse serum albumin, and porcine serum albumin, is also intended in this invention.

[0042] A disease or disability is considered "mitigated" if the severity of its symptoms, the frequency with which a patient experiences such symptoms, or both decreases.

[0043] As used herein, the terms “change,” “deletion,” “variation,” or “mutation” refer to a change in a gene present in a cell that affects the function, activity, expression (transcription or translation), or conformation of the polypeptide encoded by the gene. A change included in the present invention may be any change in a gene present in a cell that results in enhancement or disruption of the function, activity, expression, or conformation of the encoded polypeptide, including, for example, the complete absence of expression of the encoded protein, and may include missense and nonsense mutations, insertions, deletions, frameshifts, and premature termination. A change included in the present invention may alter the splicing of mRNA (splice site mutation) or cause a reading frame shift (frameshift), as stated below.

[0044] The term "amino acid sequence variant" refers to a polypeptide that has an amino acid sequence that differs to some extent from the natural sequence polypeptide. Typically, an amino acid sequence variant has at least approximately 70% homology, at least approximately 80% homology, at least approximately 90% homology, or at least approximately 95% homology to the natural polypeptide. An amino acid sequence variant has substitutions, deletions, and / or insertions at certain positions within the amino acid sequence of the natural amino acid sequence.

[0045] As used herein, the term "Ap3P" refers to adenosine-(5')-triphospho-(5')-adenosine or a salt thereof.

[0046] As used herein, the term "ARHR2" refers to autosomal recessive hypophosphatemic rickets type 2.

[0047] As used herein, the term "CKD" refers to chronic kidney disease.

[0048] As used herein, the term "CKD-MBD" refers to bone mineral metabolism disorders associated with chronic kidney disease.

[0049] As used herein, the term “coding sequence” means a sequence or part thereof of a nucleic acid or its complementary strand that can be transcribed and / or translated to produce mRNA and / or polypeptides or fragments thereof. A coding sequence includes exons in genomic DNA or immature primary RNA transcripts, which are joined together by the cell’s biochemical mechanisms to produce mature mRNA. An antisense strand is the complementary strand of such nucleic acid, from which the coding sequence can be inferred. In contrast, as used herein, the term “non-coding sequence” means a sequence or part thereof of a nucleic acid or its complementary strand that is not translated into amino acids in vivo, or into which tRNA does not interact to arrange amino acids, or does not attempt to arrange amino acids. Non-coding sequences include intron sequences in genomic DNA or immature primary RNA transcripts, and gene-related sequences, such as promoters, enhancers, and silencers.

[0050] As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., nucleotide sequences) related by the rules of base pairing. For example, the sequence “AGT” is complementary to the sequence “TCA”. Complementarity may be “partial,” in which case only some of the nucleic acid bases match according to the rules of base pairing. Alternatively, there may be “complete” or “whole” complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that depend on the binding between nucleic acids.

[0051] As used herein, the terms “composition” or “pharmaceutical composition” refer to a mixture of at least one compound useful in the present invention with a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds to patients. There are several techniques in the art for administering compounds, including, but not limited to, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, subarachnoid, gastric, ocular, pulmonary, and topical administration.

[0052] As used herein, the terms “conservative variation” or “conservative substitution” refer to the exchange of an amino acid residue with another biologically similar residue. Conservative variations or substitutions are unlikely to alter the shape of a peptide chain. Examples of conservative variations or substitutions include the exchange of one hydrophobic residue, e.g., isoleucine, valine, leucine, or methionine, with another hydrophobic residue, or the exchange of one polar residue with another polar residue, e.g., lysine to arginine, aspartic acid to glutamic acid, or asparagine to glutamine.

[0053] As used herein, the term "CUA" refers to uremic arteriolar calcification.

[0054] A "disease" is a state of health in which an animal is unable to maintain homeostasis, and if the disease does not improve, the animal's health will continue to deteriorate.

[0055] In animals, a "disorder" is a state of health in which the animal can maintain homeostasis, but the animal's health is less favorable than when the disorder is absent. Leaving a disorder untreated does not necessarily mean that the animal's health will further deteriorate.

[0056] As used herein, the term “domain” refers to a part of a molecule or structure that shares common physicochemical characteristics, including but not limited to hydrophobic, polar, spherical, and helix domains or properties. Specific examples of binding domains include, but are not limited to, DNA-binding domains and ATP-binding domains.

[0057] As used herein, the terms “effective dose,” “pharmaceutical effective dose,” and “therapeutic effective dose” refer to a quantity of a drug that is non-toxic but sufficient to produce a desired biological outcome. This outcome may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in the biological system. The appropriate therapeutic dose in any individual case can be determined by those skilled in the art using routine experimental methods.

[0058] "Code" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, that serve as a template for synthesizing other polymers and macromolecules that have a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and that have the resulting biological properties, in a biological process. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand, which is used as a template for the transcription of a gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0059] As used herein, the term "ESRD" refers to end-stage renal disease.

[0060] As used herein, the term "Fc" refers to the human IgG Fc domain. All IgG subtypes, such as IgG1, IgG2, IgG3, and IgG4, are intended to be used as Fc domains.

[0061] As used herein, the term “fragment” refers, when applied to nucleic acids, to larger subsequences of nucleic acids. A “fragment” of nucleic acids may be at least about 15 nucleotides in length, e.g., at least about 50 to about 100 nucleotides; at least about 100 to about 500 nucleotides; at least about 500 to about 1000 nucleotides; at least about 1000 to about 1500 nucleotides; about 1500 to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between). As used herein, the term “fragment” refers, when applied to proteins or peptides, to larger subsequences of proteins or peptides. A “fragment” of proteins or peptides may be at least about 20 amino acids in length, e.g., at least about 50 amino acids; at least about 100 amino acids; at least about 200 amino acids; at least about 300 amino acids; or at least about 400 amino acids (and any integer value in between).

[0062] As used herein, the term "HBD" refers to hypophosphatemic bone disease.

[0063] As used herein, the term "HHRH" refers to hereditary hypophosphatemic rickets with hypercalciuria.

[0064] "Homologousness" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. If a position in both comparison molecules is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences / the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC are 50% homologous. Generally, comparisons are performed when the two sequences are aligned to maximize homology.

[0065] As used herein, the term "IIAC" refers to idiopathic infantile arterial calcification.

[0066] As used herein, "immunoassay" refers to any binding assay that uses an antibody capable of specifically binding to a target molecule in order to detect and quantify the target molecule.

[0067] As used herein, the terms “immunoglobulin” or “Ig” are defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody found in bodily secretions, such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most targets. IgM is the most efficient immunoglobulin in agglutination, complement binding, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is unknown but which may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate-type hypersensitivity by triggering the release of mediators from mast cells and basophils when exposed to allergens.

[0068] Where the term “Educational Materials” is used herein, includes publications, records, figures, or any other medium of expression that can be used to convey the usefulness of the nucleic acids, peptides, and / or compounds of the present invention, which are included in a kit for identifying, mitigating, or treating the various diseases or disorders listed herein. Optionally, or instead, the educational materials may describe one or more methods for identifying or mitigating a disease or disorder in the cells or tissues of interest. The educational materials of the kit may, for example, be attached to a container containing the nucleic acids, polypeptides, and / or compounds of the present invention, or they may be shipped together with the container containing the nucleic acids, polypeptides, and / or compositions. Alternatively, the educational materials may be shipped separately from the container with the intention that the educational materials and compounds be used collaboratively by the recipient. Alternatively, the kit may include an applicator that can be used to administer the nucleic acids, peptides, and / or compounds of the present invention to a target. Examples of such applicators may include, for example, a drop dispenser, a bottle, a pill dispenser, a syringe, and the like.

[0069] "Isolated" means that something has been altered or removed from its natural state. For example, nucleic acids or polypeptides that are naturally present in living animals are not "isolated," but the same nucleic acids or polypeptides that have been partially or completely separated from the same material present in their natural state are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form and may exist in a non-natural environment, such as a host cell.

[0070] "Isolated nucleic acid" refers to a nucleic acid segment or fragment that is separated from adjacent sequences in its natural state, for example, a DNA fragment from which sequences normally adjacent to the DNA fragment have been removed from sequences adjacent to the DNA fragment in the naturally occurring genome. This term also applies to nucleic acids that are substantially purified from other naturally associated components, such as naturally associated RNA or DNA or proteins within a cell. Thus, this term includes recombinant DNA, for example, that is incorporated into vectors, self-replicating plasmids or viruses, or into the genomic DNA of prokaryotes or eukaryotes, or that exists as a separate molecule independent of other sequences (for example, as cDNA or genomic fragments or cDNA fragments produced by PCR or restriction enzyme digestion). This term also includes recombinant DNA that is part of a hybrid gene encoding further polypeptide sequences.

[0071] As used herein, the term "MWVC" refers to vascular wall calcification.

[0072] As used herein, the term "NPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0073] "Nucleic acid" refers to polynucleotides, including polyribonucleotides and polydeoxyribonucleotides. The nucleic acids according to the present invention may comprise any polymer or oligomer of pyrimidine bases, preferably cytosine, thymine, and uracil, and purine bases, preferably guanine (for all purposes, the whole is incorporated herein, Albert L. Lehninger, Principles of Biochemistry, 793-800 (Worth Pub. 1982)). In fact, the present invention intends any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variant thereof, such as methylated, hydroxymethylated, or glucosylated forms of these bases. These polymers or oligomers may have heterogeneous or homogeneous compositions, may be isolated from natural sources, or may be produced artificially or synthetically. Furthermore, nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

[0074] An “oligonucleotide” or “polynucleotide” is a nucleic acid having a length of at least 2 nucleotides, preferably at least 8 nucleotides, 15 nucleotides, or 25 nucleotides, but may be up to 50 nucleotides, 100 nucleotides, 1000 nucleotides, or 5000 nucleotides, and may also be a compound that specifically hybridizes to a polynucleotide. A polynucleotide includes a sequence of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or its mimetic, which may be isolated from a natural source, produced by recombination, or synthesized artificially. A further example of a polynucleotide in the present invention may be a peptide nucleic acid (PNA) (see U.S. Patent No. 6,156,501, which is incorporated herein by reference in its entirety). The present invention also encompasses situations involving unusual base pairings, such as Hoogsteen base pairings, which are specified in certain tRNA molecules and are assumed to be present in the triple helix. “Polynucleotide” and “oligonucleotide” are used synonymously herein. Where a nucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G, and C), this also includes the corresponding RNA sequence (e.g., A, U, G, C) where "U" is used instead of "T".

[0075] As used herein, the term "OPLL" refers to ossification of the posterior longitudinal ligament.

[0076] As used herein, the terms “patient,” “individual,” or “subject” refer to human or non-human mammals. Non-human mammals include, for example, livestock and pets, such as sheep, cattle, pigs, dogs, cats, and mice. For example, the patient, individual, or subject is human.

[0077] As used herein, the term “pharmaceutically acceptable” refers to a relatively non-toxic material, such as a carrier or diluent, that does not impair the biological activity or properties of a compound. That is, the material can be administered to an individual without causing undesirable biological effects or harmfully interacting with any component of a composition containing the material.

[0078] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, that is involved in transporting or delivering a pharmaceutically useful compound into or to a patient so that the compound useful in the present invention can perform its intended function. Typically, such constructs are transported or delivered from one organ or part of the body to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation containing the compound useful in the present invention and does not harm the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, e.g., cocoa butter and suppository wax; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol; polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate; agar; buffers, e.g., magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; water free of pyrogens; isotonic saline; Ringer's solution, ethyl alcohol; phosphate buffer; and other non-toxic, suitable substances used in pharmaceutical formulations. The "pharmaceutically acceptable carrier" also includes any and all coatings, antimicrobial and antifungal agents, and absorption retarders, etc., that are compatible with the activity of the compounds useful in the present invention and are physiologically acceptable to patients. Auxiliary active compounds may also be incorporated into the composition. The "pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compounds useful in the present invention.Further components that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0079] As used herein, the term “pharmaceutically acceptable salt” refers to a salt of the compound to be administered, prepared from pharmaceutically acceptable, non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, their solvates, hydrates, and inclusion compounds. A suitable pharmaceutically acceptable acid-added salt may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfates, hydrogen sulfates, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid (including monohydrogen phosphate and dihydrogen phosphate). Suitable organic acids may be selected from the aliphatic, alicyclic, aromatic, aryl-aromatic, heterocyclic, carboxyl-containing, and sulfone-containing classes of organic acids. Examples include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid. Suitable pharmaceutically acceptable base-added salts of the compounds of the present invention include, for example, alkali metal salts, alkaline earth metal salts, and transition metal salts, such as calcium salts, magnesium salts, potassium salts, sodium salts, and zinc salts. Also included are organic salts made from basic amines such as, for example, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. All of these salts can be prepared from the corresponding compounds, for example, by reacting the compounds with a suitable acid or base.

[0080] As used herein, the terms “plasma pyrophosphate level” or “plasma PPi” refer to the amount of pyrophosphate (PPi) present in the plasma of an animal. In certain embodiments, animals include, but are not limited to, mammals such as rats, mice, cats, dogs, humans, cattle, and horses. In certain embodiments, PPi is measured in plasma rather than serum because it is released from platelets. Several non-limiting methods for measuring PPi exist, one of which is a modified enzymatic assay using uridine-diphosphoglucose (UDPG) pyrophosphorylase, as described by Lust and Seegmiller (Lust, et al., 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63). Typically, healthy individuals exhibit an average plasma level of approximately 3.0 μM. Plasma PPi levels in elderly and / or calcifying or ossifying subjects are considerably lower than normal levels. In certain embodiments, subjects exhibit low plasma PPi levels of approximately 1.5 μm. In other embodiments, for subjects with calcifying disorders, plasma PPi levels are approximately 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.2 μM, 2.4 μM, and / or 2.6 μM.In other embodiments, for subjects with calcification disorders, plasma PPi levels are approximately 500 nM to 2.8 μM, 600 nM to 2.8 μM, 700 nM to 2.8 μM, 800 nM to 2.8 μM, 900 nM to 2.8 μM, 1 μM to 2.8 μM, 1.1 μM to 2.8 μM, 1.2 μM to 2.8 μM, and 1.3 μM to 2.8 μM. The concentrations are 2.8 μM, approximately 1.4 μM to 2.8 μM, approximately 1.5 μM to 2.8 μM, approximately 1.6 μM to 2.8 μM, approximately 1.7 μM to 2.8 μM, approximately 1.8 μM to 2.8 μM, approximately 1.9 μM to 2.8 μM, approximately 2 μM to 2.8 μM, approximately 2.2 μM to 2.8 μM, approximately 2.4 μM to 2.8 μM, and / or approximately 2.6 μM to 2.8 μM.

[0081] As used herein, “polynucleotide” includes cDNA, RNA, DNA / RNA hybrids, antisense RNA, ribozymes, genomic DNA, synthetic and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural, derivatized, synthetic, or semi-synthetic nucleotide bases. Modifications of wild-type or synthetic genes are also intended, including, but not limited to, deletions, insertions, substitutions, or fusions of one or more nucleotides with other polynucleotide sequences.

[0082] As used herein, the term “polypeptide” refers to a polymer compound composed of amino acid residues linked via peptide bonds, their associated native structural variants, and synthetic non-native analogs. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. As used herein, the term “protein” typically refers to a large polypeptide. As used herein, the term “peptide” typically refers to a short polypeptide. Conventional notation is used herein to represent polypeptide sequences; that is, the left end of a polypeptide sequence is an amino terminus, and the right end of a polypeptide sequence is a carboxyl terminus.

[0083] The amino acids used herein are represented by their full names, their corresponding three-letter abbreviations, or their corresponding single-letter abbreviations, as shown below: aspartic acid, Asp, D; glutamic acid, Glu, E; lysine, Lys, K; arginine, Arg, R; histidine, His, H; tyrosine, Tyr, Y; cysteine, Cys, C; asparagine, Asn, N; glutamine, Gln, Q; serine, Ser, S; threonine, Thr, T; glycine, Gly, G; alanine, Ala, A; valine, Val, V; leucine, Leu, L; isoleucine, Ile, I; methionine, Met, M; proline, Pro, P; phenylalanine, Phe, F; tryptophan, Trp, W.

[0084] As used herein, the terms “prevent” or “prevent” mean, if nothing has happened, that there will be no onset of disability or disease, or, if a disability or disease has already developed, that there will be no further onset of disability or disease. It is also considered that some or all of the symptoms associated with the disability or disease may be preventable.

[0085] As used herein, the term "PXE" refers to pseudoxanthoma elasticum.

[0086] As used herein, “sample” or “biological sample” means biological material isolated from a subject. A biological sample may contain any biological material suitable for detecting mRNA, polypeptides, or other markers of physiological or pathological processes in a subject, and may include fluids, tissues, cellular materials, and / or non-cellular materials obtained from an individual.

[0087] As used herein, "substantially purified" means essentially free from other components. For example, a substantially purified polypeptide is a polypeptide that has been isolated in its natural state from other components that would normally be associated with it.

[0088] As used herein, the terms “treatment / procedure” or “to treat / procedure” are defined as the application or administration (alone or in combination with other pharmaceutical agents) of a therapeutic agent, i.e., a compound useful in the present invention, to a patient who has a disease or disorder, has symptoms of a disease or disorder, or is at risk of developing a disease or disorder, for the purpose of curing, resolving, reducing, mitigating, altering, healing, relieving, improving, or influencing a disease or disorder, symptoms of a disease or disorder, or the likelihood of developing a disease or disorder, or the application or administration of a therapeutic agent to isolated tissue or cell lines derived from the patient (e.g., in the case of diagnostic or ex vivo application). Such treatments / procedures may be individually tailored and modified based on knowledge derived from the field of pharmacogenomics.

[0089] As used herein, the term "XLH" refers to X-linked hypophosphatemia, X-linked dominant hypophosphatemic rickets, X-linked vitamin D-resistant rickets, and / or X-linked hypophosphatemic rickets.

[0090] As used herein, the term “wild-type” refers to a gene or gene product isolated from a natural source. A wild-type gene is the one most frequently observed in a population and therefore, optionally, designated as the “normal” or “wild” type of gene. In contrast, the terms “modified” or “mutant” refer to a gene or gene product that exhibits alterations in sequence and / or functional characteristics (i.e., changes in features) compared to a wild-type gene or gene product. Natural mutants can be isolated. They are identified by the fact that they have changes in features (including changes in nucleic acid sequence) compared to a wild-type gene or gene product.

[0091] Scope: Throughout this application, various aspects of the invention can be presented in the form of a range. It should be understood that the range description is for convenience and simplification only and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the range description should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a range description such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and the individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0092] composition In certain embodiments, the polypeptide of the present invention has the formula (I): EXPORT-PROTEIN-Z-DOMAIN-XY(I), where (I) is either absent or a transport signal sequence or a biologically active fragment thereof, PROTEIN is the extracellular domain of ENPP3 (SEQ ID NO:1) or a biologically active fragment thereof, DOMAIN is selected from the group consisting of human IgG Fc domains and human albumin domains, X and Z are independently absent or polypeptides containing 1 to 20 amino acids, and Y is either absent or This is an array selected from the group consisting of TIFF0007933352000003.tif31146, where n is an integer from 1 to 20, independently for each case.

[0093] In a particular embodiment, the polypeptide comprises the extracellular domain (SEQ ID NO:1) of ENPP3 or a biologically active fragment (or region) thereof.

[0094] In one particular embodiment, the polypeptide is soluble. In another embodiment, the nuclease domain of the protein or its variant is absent. In yet another embodiment, the export is absent or selected from the group consisting of SEQ ID NO: 2 to 5. In yet another embodiment, X is absent, selected from the group consisting of a polypeptide consisting of 20 amino acids, a polypeptide consisting of 19 amino acids, a polypeptide consisting of 18 amino acids, a polypeptide consisting of 17 amino acids, a polypeptide consisting of 16 amino acids, a polypeptide consisting of 15 amino acids, a polypeptide consisting of 14 amino acids, a polypeptide consisting of 13 amino acids, a polypeptide consisting of 12 amino acids, a polypeptide consisting of 11 amino acids, a polypeptide consisting of 10 amino acids, a polypeptide consisting of 9 amino acids, a polypeptide consisting of 8 amino acids, a polypeptide consisting of 7 amino acids, a polypeptide consisting of 6 amino acids, a polypeptide consisting of 5 amino acids, a polypeptide consisting of 4 amino acids, a polypeptide consisting of 3 amino acids, a polypeptide consisting of 2 amino acids, and a polypeptide consisting of 1 amino acid. In yet another embodiment, Z is selected from the group consisting of a polypeptide consisting of 20 amino acids, 19 amino acids, 18 amino acids, 17 amino acids, 16 amino acids, 15 amino acids, 14 amino acids, 13 amino acids, 12 amino acids, 11 amino acids, 10 amino acids, 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0095] In one particular embodiment, X and Z are independently polypeptides that are absent or contain 1 to 18 amino acids. In another embodiment, X and Z are independently polypeptides that are absent or contain 1 to 16 amino acids. In yet another embodiment, X and Z are independently polypeptides that are absent or contain 1 to 14 amino acids. In yet another embodiment, X and Z are independently polypeptides that are absent or contain 1 to 12 amino acids. In yet another embodiment, X and Z are independently polypeptides that are absent or contain 1 to 10 amino acids. In yet another embodiment, X and Z are independently polypeptides that are absent or contain 1 to 8 amino acids. In yet another embodiment, X and Z are independently polypeptides that are absent or contain 1 to 6 amino acids. In yet another embodiment, X and Z are independently polypeptides that are absent or contain 1 to 5 amino acids. In yet another embodiment, X and Z are independently polypeptides that are absent or contain 1 to 4 amino acids. In yet another embodiment, X and Z are independently either absent or polypeptides containing 1 to 3 amino acids. In yet another embodiment, X and Z are independently either absent or polypeptides containing 1 to 2 amino acids. In yet another embodiment, X and Z are independently either absent or contain 1 amino acid.

[0096] In one particular embodiment, the domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the polypeptide is selected from the group consisting of SEQ ID NO: 19, 21, and 22. In yet another embodiment, the domain is a human albumin domain. In yet another embodiment, the polypeptide is selected from the group consisting of SEQ ID NO: 24, 25, and 26.

[0097] In one particular embodiment, the soluble polypeptide lacks a transmembrane domain and / or a signal peptide. In another embodiment, the soluble polypeptide lacks a transmembrane domain. In yet another embodiment, the soluble polypeptide lacks a signal peptide. In yet another embodiment, the soluble polypeptide lacks both a transmembrane domain and a signal peptide.

[0098] In one particular embodiment, the polypeptide comprises a soluble region (or fragment) of NPP3 or a fusion protein thereof, and lacks a transmembrane domain and a signal peptide. In another embodiment, the polypeptide comprises a soluble region of NPP3 and lacks a transmembrane domain and / or a signal peptide. In yet another embodiment, the polypeptide comprises a soluble region of NPP3 and lacks a transmembrane domain. In yet another embodiment, the polypeptide comprises a soluble region of NPP3 and lacks a signal peptide. In yet another embodiment, the polypeptide reduces cellular calcification when administered to subjects suffering from calcification and ossification diseases.

[0099] In one particular embodiment, the polypeptide essentially consists of SEQ ID NO:1 or a biologically active fragment thereof. In other embodiments, the polypeptide consists of SEQ ID NO:1 or a biologically active fragment thereof.

[0100] In one particular embodiment, the soluble ENPP3 fragment or its fusion protein contains the extracellular domain of ENPP3 (SEQ ID NO:1) or a biologically active fragment thereof. In another embodiment, the soluble ENPP3 fragment essentially consists of SEQ ID NO:1 or a biologically active fragment thereof. In yet another embodiment, the soluble ENPP3 fragment consists of SEQ ID NO:1 or a biologically active fragment thereof. In yet another embodiment, the soluble ENPP3 fragment or its fusion protein lacks a transmembrane domain and a signal peptide.

[0101] In certain embodiments, the polypeptide of the present invention is soluble. In other embodiments, the polypeptide of the present invention is a recombinant polypeptide. In yet another embodiment, the polypeptide of the present invention is pegylated.

[0102] method The present invention provides methods for treating or preventing diseases or disorders associated with pathological calcification or pathological ossification in subjects requiring treatment or prevention of such diseases or disorders. Furthermore, the present invention provides methods for reducing or preventing vascular calcification in subjects with low plasma pyrophosphate (PPi) levels or high serum phosphate (Pi) levels. Furthermore, the present invention provides methods for treating subjects with NPP1 deficiency or NPP1-related disorders. Furthermore, the present invention provides methods for treating or preventing disorders and diseases in subjects where an increase in the activity or level of ENPP3 polypeptide, its fragments, derivatives, variants, or variant fragments is desirable.

[0103] In one particular embodiment, a subject is administered a therapeutically effective amount of at least one polypeptide of the present invention. In another embodiment, the method includes administering a therapeutically effective amount of an isolated recombinant human soluble ENPP3 fragment or fusion protein thereof to a subject.

[0104] In certain embodiments, the disease or disorder includes at least one selected from the group consisting of GACI, IIAC, PXE, OPLL, hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic lesions, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, age-related arteriosclerosis, and calciphylaxis resulting from end-stage renal disease (or bone mineral metabolism disorders associated with chronic kidney disease).

[0105] In certain embodiments, the disease or disorder includes at least one selected from the group consisting of GACI, IIAC, PXE, OPLL, MWVC, ARHR2, ESRD, CKD-MBD, XLH, age-related osteopenia, CUA, and hypophosphatemic rickets.

[0106] In one particular aspect, the disease or disorder is GACI. In another aspect, the disease or disorder is IIAC. In yet another aspect, the disease or disorder is PXE. In yet another aspect, the disease or disorder is OPLL. In yet another aspect, the disease or disorder is hypophosphatemic rickets. In yet another aspect, the disease or disorder is osteoarthritis. In yet another aspect, the disease or disorder is calcification of atherosclerotic lesions. In yet another aspect, the disease or disorder is hereditary and non-hereditary osteoarthritis. In yet another aspect, the disease or disorder is ankylosing spondylitis. In yet another aspect, the disease or disorder is age-related arteriosclerosis. In yet another aspect, the disease or disorder is calciphylaxis (or bone mineral metabolism disorder associated with chronic kidney disease) due to end-stage renal disease. In yet another aspect, the disease or disorder is age-related osteopenia. In yet another aspect, the disease or disorder is CUA. In yet another aspect, the disease or disorder is MWVC. In yet another embodiment, the disease or disorder is ARHR2. In yet another embodiment, the disease or disorder is ESRD.

[0107] In one particular embodiment, at least one polypeptide is administered to the subject over a short or long period. In another embodiment, at least one polypeptide is administered to the subject locally, locally, or systemically. In yet another embodiment, the subject is a mammal. In yet another embodiment, the mammal is a human.

[0108] In one particular embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 250 nM. In another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 500 nM. In yet another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 800 nM. In yet another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 900 nM. In yet another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 1 μM. In yet another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 1.2 μM. In yet another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 1.4 μM. In yet another embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 1.5 μM. In a particular embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 2 μM. In a particular embodiment, the administered amount (administration of the amount) raises the plasma PPi level in the subject to at least about 4 μM.

[0109] Those skilled in the art will understand, based on the disclosures provided herein, that the present invention is useful in subjects who are being treated or are to be treated, either whole (e.g., whole-body) or partially (e.g., locally, tissue, organ) for pathological calcification or pathological ossification. In certain embodiments, the present invention is useful in the treatment or prevention of pathological calcification or pathological ossification. Those skilled in the art will recognize, based on the teachings provided herein, that the diseases and disorders treatable by the compositions and methods described herein encompass any disease or disorder in which a reduction in calcification or ossification promotes a positive therapeutic outcome.

[0110] As this disclosure, including the methods detailed herein, is obtained, it will be recognized by those skilled in the art that the present invention is not limited to the treatment of established diseases or disorders. In particular, the symptoms of the disease or disorder do not need to be present to the extent that they harm the subject. In fact, the disease or disorder does not need to be detectable in the subject before the therapeutic agent is administered. That is, there does not need to be any noticeable symptoms of the disease or disorder before the present invention may be beneficial. Accordingly, as discussed elsewhere herein, the polypeptides or variants of the present invention can be administered to a subject before the onset of a disease or disorder, thereby preventing the onset of the disease or disorder, and the present invention includes methods for preventing diseases and disorders in a subject, as will be further fully described herein.

[0111] With access to the disclosures herein, those skilled in the art will recognize that the prevention of disease or impairment in a subject includes the step of administering the polypeptide of the present invention or a variant thereof to the subject as a preventive measure against disease or impairment.

[0112] The present invention encompasses the administration of the polypeptide or its variants for carrying out the methods of the present invention. Those skilled in the art will understand, based on the disclosures provided herein, how to formulate and administer the polypeptide or its variants to a subject. However, the present invention is not limited to any particular method of administration or treatment regimen. This is especially true when, given the disclosures provided herein, including reduction to practice using models of pathological calcification or pathological ossification recognized in the art, it is recognized by those skilled in the art that the method of administering the compounds of the present invention can be determined by those skilled in the pharmacological field.

[0113] Pharmaceutical compositions and formulations The present invention intends to use a pharmaceutical composition containing the polypeptide of the present invention in the method of the present invention.

[0114] Such pharmaceutical compositions take a form suitable for administration to a target. Alternatively, the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers, one or more further components, or any combination thereof. The various components of the pharmaceutical composition may exist in the form of physiologically acceptable salts, for example, in combination with physiologically acceptable cations or anions, as is well known in the art.

[0115] In certain embodiments, a pharmaceutical composition useful for carrying out the method of the present invention may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In other embodiments, a pharmaceutical composition useful for carrying out the present invention may be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0116] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any further components in the pharmaceutical composition of the present invention vary depending on the identity, size, and condition of the subject being treated, and further, depending on the route through which the composition is administered. For example, the composition may contain about 0.1% to about 100% (w / w) of the active ingredient.

[0117] Pharmaceutical compositions useful in the method of the present invention may be developed as appropriate for administration via inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, subarachnoid, intravenous, or other routes. Other intended formulations include injection nanoparticles, liposome preparations, reencapsulated red blood cells containing active ingredients, and immunotherapy formulations. The route of administration is readily apparent to those skilled in the art and depends on any number of factors, including the type and severity of the disease being treated, and the type and age of the veterinary animal or human patient being treated.

[0118] Formulations of the pharmaceutical compositions described herein may be prepared by any method known in the field of pharmacology, or by any method to be developed in the field of pharmacology in the future. Generally, such preparation methods include the steps of conjugating the active ingredient with a carrier or one or more other accessory components, and then, if necessary or desired, forming or packaging the product into a desired single-dose unit or multi-dose unit.

[0119] As used herein, “unit dose” refers to a separate amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose administered to the subject, or a convenient portion of such a dose, for example, half or one-third of such a dose. The unit dosage form may be a single-dose unit dosage form or one unit dosage form for multiple daily doses (e.g., about 1 to 4 times or more per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0120] The descriptions of pharmaceutical compositions provided herein are primarily directed toward pharmaceutical compositions suitable for ethical administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to all types of animals. It is well understood that pharmaceutical compositions suitable for administration to humans can be modified to make them suitable for administration to various animals, and such modifications can be designed and carried out by a veterinary pharmacologist with ordinary knowledge, if any, with ordinary experiments. The subjects to which the pharmaceutical compositions of the present invention are intended to be administered include, but are not limited to, humans and other primates, commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, and dogs.

[0121] In certain embodiments, the composition is formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the active agent and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphates and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are listed in Remington's Pharmaceutical Sciences, 1991, Mack Publication Co., New Jersey.

[0122] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained by using coatings such as lecithin to maintain the required particle size in the case of dispersions, and by using surfactants. Microbial activity can be inhibited by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Often, it is preferable that the composition contains isotonic agents, such as sugars, sodium chloride, or polyhydric alcohols, such as mannitol and sorbitol. Long-term absorption of the injectable composition is made possible by including absorption retarders, such as aluminum monostearate and gelatin, in the composition.

[0123] The formulation may be used in combination with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carriers suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable method of administration known to those skilled in the art. The pharmaceutical preparations may be sterilized and, if desired, may be mixed with adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavorings, and / or aromatics. These may, if desired, be combined with other active agents, such as other analgesics.

[0124] The “further components” as used herein include, but are not limited to, excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions, e.g., gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, lubricants; buffers; salts; thickeners; bulking agents; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and one or more pharmaceutically acceptable polymeric or hydrophobic materials. Other “further components” that may be included in the pharmaceutical compositions of the present invention are known in the art, for example, as described by reference herein, Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0125] The compositions of the present invention may contain about 0.005% to 2.0% of a preservative based on the total weight of the composition. The preservative is used to prevent degradation when exposed to pollutants in the environment. Examples of useful preservatives according to the present invention include, but are not limited to, preservatives selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidourea, and combinations thereof. Particularly preferred preservatives are combinations of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

[0126] The composition preferably contains antioxidants and chelating agents that inhibit the degradation of the compounds. Preferred antioxidants for some compounds are BHT, BHA, α-tocopherol, and ascorbic acid in a preferred range of about 0.01% to 0.3% by weight of the total weight of the composition, more preferably BHT in a range of 0.03% to 0.1% by weight. Preferably, the chelating agent is present in an amount of 0.01% to 0.5% by weight of the total weight of the composition. Particularly preferred chelating agents include EDTA salts (e.g., disodium EDTA) and citric acid in a weight range of about 0.01% to 0.20% by weight of the total weight of the composition, more preferably 0.02% to 0.10% by weight. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. BHT and disodium edetate are particularly preferred antioxidants and chelating agents for certain compounds, respectively, but other suitable and equivalent antioxidants and chelating agents may be substituted, as are known to those skilled in the art.

[0127] Liquid suspensions can be prepared using conventional methods of suspending the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, tonsil oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further include, but are not limited to, one or more further components, such as suspending agents, dispersants or wetting agents, emulsifiers, lubricants, preservatives, buffers, salts, flavorings, colorants, and sweeteners. Oily suspensions may further include thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum, and cellulose derivatives (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose, methylcellulose). Known dispersants or wetting agents include, but are not limited to, natural phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, condensation products of alkylene oxides with long-chain aliphatic alcohols, condensation products of alkylene oxides with partial esters derived from fatty acids and hexitol, or condensation products of alkylene oxides with partial esters derived from fatty acids and hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and gum arabic. Known preservatives include, but are not limited to, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, or n-propyl parahydroxybenzoate, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickeners for oily suspensions include, for example, beeswax, solid paraffin, and cetyl alcohol.

[0128] Liquid solutions of active ingredients dissolved in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, “oily” liquids are liquids that contain carbon-containing liquid molecules and exhibit characteristics of being less polar than water. Liquid solutions of the pharmaceutical compositions of the present invention may contain each of the components described for liquid suspensions, and it is understood that the suspending agent does not necessarily help dissolve the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic salines. Oily solvents include, for example, tonsil oil, oily esters, ethyl alcohol, vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0129] Powdered and granular formulations of the pharmaceutical composition of the present invention can be prepared using known methods. Such formulations may be administered directly to a subject, or they may be used, for example, to form tablets, to fill capsules, or to prepare aqueous or oily suspensions or solutions by adding aqueous or oily vehicles. Each of these formulations may further contain one or more dispersants or wetting agents, suspending agents, and preservatives. These formulations may also contain further excipients such as bulking agents and sweeteners, flavorings, or coloring agents.

[0130] The pharmaceutical compositions of the present invention may also be prepared, packaged, or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oil phase may be a vegetable oil, e.g., olive oil or peanut oil, a mineral oil, e.g., liquid paraffin, or a combination thereof. Such compositions may further contain one or more emulsifiers, e.g., natural rubber, e.g., gum arabic or tragacanth gum, natural phosphatides, e.g., soy phosphatide or lecithin phosphatide, esters or partial esters derived from a combination of fatty acids and hexitol anhydride, e.g., sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, e.g., polyoxyethylene sorbitan monooleate. These emulsions may also contain further components, e.g., sweeteners or flavorings.

[0131] Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods for adhering or bonding chemical compositions to a surface, methods for incorporating chemical compositions into the structure of a material during the synthesis of the material (i.e., together with physiologically degradable materials), and methods for absorbing aqueous or oily solutions or suspensions into an absorbent material, with or without subsequent drying.

[0132] Administration / Medication The administration plan may affect what constitutes the effective dose. For example, several divided doses and staggered doses may be administered daily or consecutively. Alternatively, the dose may be administered by continuous infusion or as a bolus injection. Furthermore, the dose of the therapeutic formulation may be increased or decreased proportionally, as indicated by the urgent requirements of the treatment or prevention situation.

[0133] The compositions of the present invention can be administered to patients, preferably mammals, more preferably humans, in doses and durations effective for treating a disease or disorder in the patient, using known procedures. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound used, the time of administration, the elimination rate of the compound, the duration of treatment, other drugs, compounds, or materials used in combination with the compound, the state of the disease or disorder, the age, sex, weight, condition, overall health, and medical history of the patient being treated, as well as similar factors well known in the medical field. The administration plan may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgent requirements of the treatment situation. A non-limiting example of the effective dose range of the therapeutic compounds of the present invention is about 0.01 to 50 mg / kg body weight / day. Those skilled in the art will be able to study the relevant factors and make a determination regarding the effective amount of the therapeutic compound without excessive experimentation.

[0134] The compound may be administered to animals several times a day, or less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or once a year. The amount of the compound administered per day may, in non-limiting examples, be administered daily, every other day, every two days, every three days, every four days, or every five days. For example, when administered every other day, a dose of 5 mg per day may be started on Monday, followed by a first dose of 5 mg per day on Wednesday, and a second dose of 5 mg per day on Friday. The frequency of administration is readily apparent to those skilled in the art and depends on any number of factors, including, but not limited to, the type and severity of the disease being treated, as well as the type and age of the animal.

[0135] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be modified to obtain an amount of the active ingredient that is non-toxic to the patient and effective in achieving a desired therapeutic response for a particular patient, composition, and method of administration.

[0136] A medical doctor with ordinary knowledge in the art, such as a physician or veterinarian, can easily determine the effective amount of the required pharmaceutical composition and prescribe that effective amount. For example, a physician or veterinarian can start administering the compound of the present invention used in the pharmaceutical composition at a level lower than necessary to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0137] In certain embodiments, it is particularly advantageous to formulate compounds in unit dosage forms to facilitate administration and to ensure uniformity of dosage. As used herein, a unit dosage form refers to a physically distinct unit suitable as a unit dose to a patient to be treated. Each unit contains a predetermined amount of the therapeutic compound calculated to bind to the required pharmaceutical vehicle to produce the desired therapeutic effect. The unit dosage forms of the present invention are determined and directly depended on (a) the unique characteristics of the therapeutic compound and the specific therapeutic effect to be achieved, and (b) the constraints inherent in the field in which such therapeutic compounds are formulated / formulated to treat a disease or disorder in a patient.

[0138] In certain embodiments, the compositions of the present invention are administered to patients in doses of 1 to 5 times a day or more. In other embodiments, the compositions of the present invention are administered to patients in a range of dosing, including, but not limited to, once a day, once every two days, once every three days, once a week, and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present invention will differ from subject to subject, depending on many factors, including, but not limited to, age, the disease or disorder being treated, sex, overall health, and other factors. Therefore, the present invention should not be construed as being limited to any particular dosing schedule, and the exact dosage and composition to be administered to any patient will be determined by the attending physician, taking into account all other factors concerning the patient.

[0139] The compounds of the present invention for administration are approximately 1 μg to 7,500 mg, approximately 20 μg to 7,000 mg, approximately 40 μg to 6,500 mg, approximately 80 μg to 6,000 mg, approximately 100 μg to 5,500 mg, approximately 200 μg to 5,000 mg, approximately 400 μg to 4,000 mg, approximately 800 μg to 3,000 mg, approximately 1 mg to 2,500 mg, and approximately 2 mg to 2,000 mg. mg, approximately 5 mg to approximately 1,000 mg, approximately 10 mg to approximately 750 mg, approximately 20 mg to approximately 600 mg, approximately 30 mg to approximately 500 mg, approximately 40 mg to approximately 400 mg, approximately 50 mg to approximately 300 mg, approximately 60 mg to approximately 250 mg, approximately 70 mg to approximately 200 mg, approximately 80 mg to approximately 150 mg, and any and all total or partial increases in between.

[0140] In some embodiments, the dose of the compound of the present invention is about 0.5 μg to about 5,000 mg. In some embodiments, the dose of the compound of the present invention used in the compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, as well as any and all of the total or partial increases thereof.

[0141] In certain embodiments, the present invention relates to a packaged pharmaceutical composition comprising a container for containing a therapeutically effective amount of the compound of the present invention, either alone or in combination with a second pharmaceutical agent, and instructions for using the compound in a patient to treat, prevent, or reduce one or more symptoms of a disease or disorder.

[0142] The term “container” includes any container for housing a pharmaceutical composition. For example, in certain embodiments, the container is a packaging container for holding a pharmaceutical composition. In other embodiments, the container is not a packaging container for holding a pharmaceutical composition. That is, the container is a container, such as a box or vial, for holding a packaged or unpackaged pharmaceutical composition and instructions for using the pharmaceutical composition. Furthermore, packaging techniques are well known in the art. It should be understood that instructions for using a pharmaceutical composition may be placed on the surface of the packaging container for holding the pharmaceutical composition. Thus, the instructions establish an enhanced functional relationship with the packaged product. However, it should be understood that the instructions may contain information about the compound’s ability to perform its intended function, for example, to treat, prevent, or reduce a disease or disorder in a patient.

[0143] Route of administration Routes of administration of any composition of the present invention include inhalation, oral administration, nasal administration, rectal administration, parenteral administration, sublingual administration, transdermal administration, transmucosal administration (e.g., sublingual administration, lingual administration, (trans)chuccal administration, (trans)urethral administration, vaginal administration (e.g., transvaginal and perivaginal administration), nasal (intranasal) administration, and (trans)rectal administration), intravesical administration, intrapulmonary administration, intraduodenal administration, intragastric administration, subarachnoid administration, subcutaneous administration, intramuscular administration, intradermal administration, intra-arterial administration, intravenous administration, intrabronchial administration, inhalation administration, and topical administration.

[0144] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magma preparations, lozenges, creams, pastes, ointments, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powders or aerosolized preparations for inhalation, and compositions and preparations for intravesical administration. It should be understood that the formulations and compositions useful in the present invention are not limited to the specific formulations and compositions described herein.

[0145] Oral administration For oral administration, tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel caps are particularly suitable. Other formulations suitable for oral administration include, but are not limited to, powders or granules, aqueous or oily suspensions, aqueous or oily solutions, pastes, gels, toothpastes, mouthwashes, coatings, oral rinses, or emulsions. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents, e.g., lactose; granulators and disintegrants, e.g., corn starch; binders, e.g., starch; and lubricants, e.g., magnesium stearate.

[0146] The tablets do not need to be coated, but may be coated using known methods to slow their disintegration in the target gastrointestinal tract, thereby allowing for sustained release and absorption of the active ingredient. For example, materials such as glyceryl monostearate or glyceryl distearate may be used to coat the tablets. Furthermore, for example, the tablets may be coated using the methods described in U.S. Patents No. 4,256,108; No. 4,160,452; and No. 4,265,874 to form osmotically controlled release tablets. The tablets may further contain sweeteners, flavorings, colorings, preservatives, or any combination thereof to provide a pharmaceutically excellent and palatable preparation.

[0147] Hard capsules containing the active ingredient may be made using a physiologically biodegradable composition such as gelatin. Such hard capsules contain the active ingredient and may further contain additional components, such as an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin.

[0148] Soft gelatin capsules containing the active ingredient may be made using a physiologically biodegradable composition such as gelatin. Such soft capsules contain the active ingredient, which may be mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0149] For oral administration, the compounds of the present invention may take the form of tablets or capsules prepared by conventional means using pharmaceutically acceptable excipients, such as binders; fillers; lubricants; disintegrants; or wetting agents. If desired, the tablets may be coated using appropriate methods and coating materials, such as the OPADRY® film coating system available from Colorcon, West Point, Pa (e.g., OPADRY® OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type, and OPADRY® White, 32K18400).

[0150] Liquid preparations for oral administration may take the form of solutions, syrups, or suspensions. Liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fat); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., tonsil oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl parahydroxybenzoate, propyl parahydroxybenzoate, or sorbic acid). Liquid formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged, or sold in liquid form, or in the form of a dry product intended for reconstitution with water or another suitable vehicle before use.

[0151] Tablets containing an active ingredient may be made, for example, by compressing or molding the active ingredient together with one or more additional ingredients, if optional. Compressed tablets may be prepared by compressing a freely flowable active ingredient, such as a powder or granular preparation, with one or more binders, lubricants, excipients, surfactants, and dispersants, if optional, in a suitable machine. Wet tablets may be prepared by molding a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least enough liquid to moisten the mixture, in a suitable machine. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulators and disintegrants, binders, and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, crystalline cellulose, calcium phosphate, monocalcium phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pre-gelatinized corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

[0152] Granulation is a well-known method in the pharmaceutical field for modifying starting powders or other particulate materials of active ingredients. This powder is typically mixed with a binder material to form larger, more durable, and freely flowable aggregates or granules, known as "granulations." For example, a solvent-based "wet" granulation process generally involves mixing the powder with a binder material, moistening it with water or an organic solvent under conditions that form a wet granular mass, and then evaporating the solvent from the wet granular mass.

[0153] Generally, melt granulation essentially involves using a material that is solid or semi-solid at room temperature (i.e., has a relatively low softening or melting point range) to facilitate the granulation of powdered materials or other materials in the absence of added water or other liquid solvents. When heated to a temperature within its melting point range, the low-melting-point solid liquefies and acts as a binder or granulation medium. The liquefied solid spreads as is over the surface of the powdered material it is in contact with, and as it cools, forms a solid granular mass to which the initial material is bound. The resulting melt granules may then be fed into a tablet press or encapsulated to prepare oral dosage forms. Melt granulation improves the dissolution rate and bioavailability of the active ingredient (i.e., drug) by forming a solid dispersion or solid solution.

[0154] U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when the wax, in a molten state, is mixed with a specific flow-improving additive, and the mixture is subsequently cooled and granulated. In certain embodiments, the wax itself only melts when the wax and additive are melted and combined, while in other embodiments, both the wax and the additive melt.

[0155] The present invention also includes a multilayer tablet comprising a layer that delays the release of one or more compounds useful in the method of the present invention, and a further layer that immediately releases one or more compounds useful in the method of the present invention. Using a wax / pH-sensitive polymer mixture, a stomach-insoluble composition can be obtained that encapsulates the active ingredient and ensures the delayed release of the active ingredient.

[0156] Parenteral administration As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by creating a physical hole in the target tissue and administering the pharmaceutical composition through the hole in the tissue. Therefore, parenteral administration includes, but is not limited to, the administration of a pharmaceutical composition by injection, application of a pharmaceutical composition through a surgical incision, or application of a pharmaceutical composition through a non-surgical wound penetrating the tissue. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and renal dialysis infusion.

[0157] Formulations of pharmaceutical compositions suitable for parenteral administration include an active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for large-volume instantaneous or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage forms, for example, in ampoules or in multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions dissolved in oily or aqueous vehicles, paste formulations, and portable sustained-release formulations or biodegradable formulations. Such formulations may further include, but are not limited to, one or more further components, including suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry form (i.e., powder or granules) for reconstitution of the reconstituted composition using a suitable vehicle (e.g., sterile, pyrogen-free water) before parenteral administration.

[0158] Pharmaceutical compositions may be prepared, packaged, or sold in the form of sterile aqueous or oily suspensions or solutions for injection. These suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, further components such as dispersants, wetting agents, or suspending agents as described herein. Such sterile injection formulations may be prepared using non-toxic, parenterally acceptable diluents or solvents, e.g., water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solutions, and non-volatile oils, e.g., synthetic monoglycerides or diglycerides. Other useful parentally administered formulations include formulations containing the active ingredient in the form of microcrystalline material, encapsulated in liposome preparations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, e.g., emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

[0159] Further forms of administration Further dosage forms of the present invention include those described in U.S. Patent Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Further dosage forms of the present invention also include those described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Further dosage forms of the present invention also include those described in PCT applications WO03 / 35041, WO03 / 35040, WO03 / 35029, WO03 / 35177, WO03 / 35039, WO02 / 96404, WO02 / 32416, WO01 / 97783, WO01 / 56544, WO01 / 32217, WO98 / 55107, WO98 / 11879, WO97 / 47285, WO93 / 18755, and WO90 / 11757.

[0160] Sustained-release formulations and drug delivery systems Sustained-release or sustained-release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques. In some cases, the dosage form used may be provided to slowly release or sustainably release one or more active ingredients contained therein, for example, by using hydropropylmethyl cellulose, other polymer matrices, gels, dialysis membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres, or a combination thereof, in different ratios to provide a desired release profile. Suitable sustained-release formulations known to those skilled in the art, including the sustained-release formulations described herein, can be readily selected for use with the pharmaceutical compositions of the present invention. Accordingly, single-unit dosage forms suitable for oral administration and adapted for sustained release, such as tablets, capsules, gel caps, and caplets, are included in the present invention.

[0161] Most sustained-release pharmaceutical products share the common goal of improving pharmacotherapy beyond what can be achieved with uncontrolled counterparts. Ideally, the use of optimally designed sustained-release formulations in medical procedures is characterized by the use of the smallest possible amount of active pharmaceutical ingredient to cure or control a condition in the shortest possible time. Advantages of sustained-release formulations include prolonged drug activity, reduced administration frequency, and high patient compliance. Furthermore, sustained-release formulations can be used to influence the onset time of action or other characteristics, such as the blood concentration of the drug, and therefore the occurrence of side effects.

[0162] Most sustained-release formulations are designed to initially release a rapid amount of the drug to produce the desired therapeutic effect, and then gradually and continuously release other amounts of the drug to maintain this level of therapeutic effect over a longer period. In order to maintain this constant drug level in the body, the drug must be released from the formulation at a rate that compensates for the amount of drug being metabolized and excreted from the body.

[0163] The sustained release of the active ingredient can be stimulated by various inductive factors, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. In the context of the present invention, the term "sustained-release ingredient" is defined herein as a compound that facilitates the sustained release of the active ingredient, including but not limited to polymers, polymer matrices, gels, dialysis membranes, liposomes, or microspheres, or combinations thereof.

[0164] In certain embodiments, the formulations of the present invention may be short-acting formulations, rapid-offset formulations, and sustained-release formulations, such as sustained-release formulations, delayed-release formulations, and pulsatile-release formulations, but are not limited thereto.

[0165] The term "sustained-release" is used in its conventional sense to refer to a drug formulation that gradually releases a drug over a long period of time, and may, but may not necessarily, result in a substantially constant drug blood concentration over a long period. The period may be longer than one month and should be longer than the release of an equivalent amount of drug administered in bolus form. In the case of sustained-release, the compound may be formulated with a suitable polymer or hydrophobic material that imparts sustained-release properties to the compound. Accordingly, the compound for use in the method of the present invention may be administered in the form of microparticles, for example by injection, or by implantation in the form of a cachet or disc. In a preferred embodiment of the present invention, the compound of the present invention is administered to the patient alone or in combination with another pharmaceutical agent using a sustained-release formulation.

[0166] The term "delayed release" is used herein in its conventional sense to refer to a drug formulation that releases the drug for the first time after administration, after a delay of some time, which may include a delay of approximately 10 minutes to approximately 12 hours, although this is not necessarily the case. The term "pulsatile release" is used herein in its conventional sense to refer to a drug formulation that releases the drug after administration in such a way that it produces a drug-pulsed plasma profile. The term "immediate release" is used herein in its conventional sense to refer to a drug formulation that releases the drug immediately after administration.

[0167] As used herein, "short-term" means any period of time, including, up to, or including any, all, total or partial increments thereof, up to, approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 40 minutes, 20 minutes, or 10 minutes after the administration of the drug.

[0168] As used herein, rapid elimination means any period of time, including, up to, approximately 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 40 minutes, 20 minutes, or 10 minutes, after the administration of the drug, and any and all of its total and partial increases.

[0169] Those skilled in the art will recognize, or can recognize by routine experimental methods alone, a great many equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the appended claims. For example, changes in reaction conditions, including but not limited to reaction time, reaction size / volume, and experimental reagents, such as solvents, catalysts, pressure, atmospheric conditions, such as nitrogen atmosphere, and reducing / oxidizing agents, by alternatives recognized in the art and by routine experimental methods alone, should be understood to be within the scope of this application.

[0170] Wherever values ​​and ranges are provided herein, it should be understood that all values ​​and ranges contained therein are intended to be within the scope of the present invention. Furthermore, all values ​​within these ranges, as well as any upper or lower limits on the ranges of values, are also included in this application.

[0171] The following embodiments further illustrate aspects of the present invention. However, the following embodiments do not limit the teachings or disclosures of the present invention set forth herein. [Examples]

[0172] The present invention will now be described in relation to the following examples. These examples are provided for illustrative purposes only. The present invention is not limited to these examples and includes all variations that are evident as a result of the teachings provided herein.

[0173] Method and materials: Arrangement: Extracellular domain of ENPP3 (SEQ ID NO:1) TIFF0007933352000004.tif88139 signal sequence ENPP7 (SEQ ID NO:2) TIFF0007933352000005.tif3128 Signal sequence ENPP7 (SEQ ID NO:3) TIFF0007933352000006.tif3128 Signal sequence ENPP5 (SEQ ID NO:4) TIFF0007933352000007.tif3128 Signal sequence ENPP1-2-1 (SEQ ID NO: 5) TIFF0007933352000008.tif25143 (single underline)-(double underline): A residue that has been exchanged with residues 1-27 of NPP2 so that it is cleaved at the point where it changes from a single underline to a double underline. SEQ ID NO:6 (DSS) n In the array, n is an integer between 1 and 20. SEQ ID NO:7 (ESS) n In the array, n is an integer between 1 and 20. SEQ ID NO:8 (RQQ) n In the array, n is an integer between 1 and 20. SEQ ID NO:9 (KR) n In the array, n is an integer between 1 and 20. SEQ ID NO:10 R n In the array, n is an integer between 1 and 20. SEQ ID NO:11 (KR) n In the array, n is an integer between 1 and 20. TIFF0007933352000009.tif57128SEQ ID NO:17 En In the array, n is an integer between 1 and 20. SEQ ID NO:18 D n In the array, n is an integer between 1 and 20.

[0174] ENPP121-NPP3-Fc sequence (SEQ ID NO: 19) TIFF0007933352000010.tif118139 Bold residues = amino acid sequences derived from NPP1; single underlined residues = signal peptide sequences derived from NPP2; double underlined residues = amino acid sequences of the IgG Fc domain. In certain embodiments, the IgG Fc domain is selected from any of the subclasses IgG1, IgG2, IgG3, and IgG4. In other embodiments, an albumin domain is used instead of the Fc domain.

[0175] In one particular embodiment, the NPP3 C-terminus and Fc / albumin domain are linked by a linker. In another embodiment, the linker contains at least two amino acids. In yet another embodiment, the linker contains 2 to 40 amino acids, 2 to 30 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 16 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 2 to 4 amino acids, or 2 amino acids. In yet another embodiment, the mobile linker contains polyethylene glycol chains and / or hydrocarbon chains (e.g., alkylene chains).

[0176] IgG Fc sequence (SEQ ID NO:20) TIFF0007933352000011.tif21145

[0177] ENPP7-NPP3-Fc sequence (SEQ ID NO:21) TIFF0007933352000012.tif112139 Single underlined residues = signal peptide sequence derived from NPP7; double underlined residues = amino acid sequence of the IgG Fc domain. In certain embodiments, the IgG Fc domain is selected from any of the subclasses IgG1, IgG2, IgG3, and IgG4. In other embodiments, an albumin domain is used instead of the Fc domain.

[0178] In one particular embodiment, the NPP3 C-terminus and Fc / albumin domain are linked by a linker. In another embodiment, the linker contains at least two amino acids. In yet another embodiment, the linker contains 2 to 40 amino acids, 2 to 30 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 16 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 2 to 4 amino acids, or 2 amino acids. In yet another embodiment, the mobile linker contains polyethylene glycol chains and / or hydrocarbon chains (e.g., alkylene chains).

[0179] ENPP5-NPP3-Fc sequence (SEQ ID NO:22) TIFF0007933352000013.tif112139 Single underlined residues = signal peptide sequence derived from NPP5; double underlined residues = amino acid sequence of the IgG Fc domain. In certain embodiments, the IgG Fc domain is selected from any of the subclasses IgG1, IgG2, IgG3, and IgG4. In other embodiments, an albumin domain is used instead of the Fc domain.

[0180] In one particular embodiment, the NPP3 C-terminus and Fc / albumin domain are linked by a linker. In another embodiment, the linker contains at least two amino acids. In yet another embodiment, the linker contains 2 to 40 amino acids, 2 to 30 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 16 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 2 to 4 amino acids, or 2 amino acids. In yet another embodiment, the mobile linker contains polyethylene glycol chains and / or hydrocarbon chains (e.g., alkylene chains).

[0181] Albumin sequence (SEQ ID NO:23) TIFF0007933352000014.tif57145

[0182] ENPP121-NPP3-albumin sequence (SEQ ID NO:24) TIFF0007933352000015.tif160139 Bold residues = amino acid sequence derived from NPP1; single underlined residues = signal peptide sequence derived from NPP2; double underlined residues = spacer sequence and amino acid sequence of the albumin domain.

[0183] In one particular embodiment, the NPP3 C-terminus and albumin domain are linked by a linker. In another embodiment, the linker contains at least two amino acids. In yet another embodiment, the linker contains 2 to 40 amino acids, 2 to 30 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 16 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 2 to 4 amino acids, or 2 amino acids. In yet another embodiment, the mobile linker contains polyethylene glycol chains and / or hydrocarbon chains (e.g., alkylene chains).

[0184] ENPP7-NPP3-albumin sequence (SEQ ID NO:25) TIFF0007933352000016.tif154139 Single underlined residues = signal peptide sequence derived from NPP7; double underlined residues = spacer sequence and amino acid sequence of albumin domain.

[0185] In one particular embodiment, the NPP3 C-terminus and albumin domain are linked by a linker. In another embodiment, the linker contains at least two amino acids. In yet another embodiment, the linker contains 2 to 40 amino acids, 2 to 30 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 16 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 2 to 4 amino acids, or 2 amino acids. In yet another embodiment, the mobile linker contains polyethylene glycol chains and / or hydrocarbon chains (e.g., alkylene chains).

[0186] ENPP5-NPP3-albumin sequence (SEQ ID NO:26) TIFF0007933352000017.tif154139 Single underlined residues = signal peptide sequence derived from NPP5; double underlined residues = spacer sequence and amino acid sequence of albumin domain.

[0187] In one particular embodiment, the NPP3 C-terminus and albumin domain are linked by a linker. In another embodiment, the linker contains at least two amino acids. In yet another embodiment, the linker contains 2 to 40 amino acids, 2 to 30 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 16 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 2 to 4 amino acids, or 2 amino acids. In yet another embodiment, the mobile linker contains polyethylene glycol chains and / or hydrocarbon chains (e.g., alkylene chains).

[0188] Nucleotide sequence of NPP121-NPP3-Fc (SEQ ID NO: 27) TIFF0007933352000018.tif208128TIFF0007933352000019.tif135128

[0189] Nucleotide sequence of NPP121-NPP3-Fc (SEQ ID NO: 28) TIFF0007933352000020.tif69128TIFF0007933352000021.tif220128TIFF0007933352000022.tif172128

[0190] Nucleotide sequence of hNPP3-hFc-pcDNA3 (SEQ ID NO:29) TIFF0007933352000023.tif33128TIFF0007933352000024.tif220128TIFF00079333520 00025.tif220128TIFF0007933352000026.tif220128TIFF0007933352000027.tif184128

[0191] Example 1: Figures 1A-1C include graphs illustrating studies on the steady-state ATP hydrolysis activity of hNPP3.

[0192] As illustrated in Figure 1A, the time course of AMP product formation was analyzed after adding 0.98 μM, 1.95 μM, 3.9 μM, 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM ATP (from bottom to top) to 50 nM hNPP3. The enzymatic reaction was stopped at various time points with equal amounts of 3 M formic acid, and the reaction product AMP was quantified by HPLC analysis using an AMP calibration curve. A flat line passing through the data points best fit the nonlinear enzyme kinetic model with product inhibition and substrate depletion.

[0193] Figure 1B shows the steady state. at A comparison of ATPase turnover rates is shown in the figure. Initial steady state. atThe substrate concentration dependence of the enzyme turnover rate of hNPP3 was compared with that measured for hNPP1. Both the ATPase cycling reactions of 50 nM hNPP3 and hNPP1 depleted the ATP substrate within 1 minute at 0.98 μM, 1.95 μM, and 3.9 μM ATP. The uncertainties at these low ATP concentrations were quite large, therefore these three rates were excluded from the dataset during fitting. hNPP3 The fixed steady state at The ATPase reaction reached a maximum value of 2.59(±0.04) s from the weighted average of the rate measurements at 7.8 μM, 15.6 μM, 31.3 μM, 62.5 μM, and 125 μM substrate -1 enzyme -1 (k cat ). The turnover rate of hNPP1 was 3.46(±0.44) s -1 enzyme -1 . The K M for the ATP substrate was estimated to be <8 μM.

[0194] Figure 1C illustrates the substrate concentration dependence of the η value. Since the η value decreases with increasing substrate concentration for both enzymes, it can be seen that the non-linearity in the progress of the enzyme reaction is contributed far more by substrate depletion than by product inhibition at low initial substrate concentrations. The similarity of the η values of hNPP3 and hNPP1 is consistent, and the reaction rates and product inhibition of the two enzymes are similar.

[0195] Example 2: Animal Model The following non-limiting animal models can be used to test the efficacy of the presently claimed compositions against human diseases caused by low pyrophosphate (PPi).

[0196] 1. enpp1asj / asj model of generalized arterial calcification of infancy (GACI); Li, et al., 2013, Disease Models & Mech. 6(5):1227-35. 2. enpp12asj / 2asj model of generalized arterial calcification of infancy (GACI); Li, et al., 2014, PloS one 9(12):e113542. 3. ABCC6- / - mouse model of pseudoxanthoma elasticum (PXE); Jiang, et al., 2007, J. Invest. Derm. 127(6):1392-402. 4. A mouse model of HYP (hypophosphatemic dysphosphate) of X-linked hypophosphatemia (XLH); Liang, et al., 2009, Calcif. Tissue Int. 85(3):235-46. 5. LmnaG609G / + mouse model of Hutchison-Gilford Progeria Syndrome; Villa-Bellosta, et al., 2013, Circulation 127(24):2442-51. 6. Tip-toe walking (ttw) mouse models of ossification of the posterior longitudinal ligament (OPLL) (Okawa, et al., 1998, Nature Genetics 19(3):271-3; Nakamura, et al., 1999, Human Genetics 104(6):492-7) and osteoarthritis (Bertrand, et al., 2012, Annals Rheum. Diseases 71(7):1249-53) 7. A rat model of chronic kidney disease (CKD) fed an adenine-based diet; Schibler, et al., 1968, Clin. Sci. 35(2):363-72; O'Neill, et al., 2011, Kidney Int. 79(5):512-7. 8. A mouse model of chronic kidney disease (CKD) fed an adenine-rich diet; Jia, et al., 2013, BMC Nephrol. 14:116. 9. A rat model of CKD after 5 / 6th nephrectomy; Morrison, 1962, Lab Invest. 11:321-32; Shimamura & Morrison, 1975, Am. J. Pathol. 79(1):95-106. 10. ENPP1 knockout mouse models for GACI and osteopenia; Mackenzie, et al., 2012, PloS one 7(2):e32177.

[0197] In certain aspects, there are no rodent models that reproduce adult-onset human disease GACI, also known in the literature as autosomal recessive hypophosphatemic rickets type 2 (ARHR2) (Levy-Litan, et al., 2010, Am. J. Human Gen. 86(2):273-8).

[0198] Experimental details regarding enzyme activity, quantification of plasma PPi, micro-CT scans, quantification of plasma pyrophosphate uptake, and a mouse calcification model are described in patent applications and / or publications PCT / US2016 / 33236, WO2014126965 (related to PCT patent application number PCT / US2014 / 015945), and US20150359858, respectively. These are each incorporated herein by reference in their entirety.

[0199] Example 3: Preparation and purification of ENPP3 fusion protein ENPP3 is produced by establishing stable transfection in either CHO or HEK293 mammalian cells. This protein can be produced in either adherent or suspension cells. To establish stable cell lines, nucleic acid sequences encoding the NPP3 fusion protein (Figures 3-5 and SEQ ID NO: 1-29) were introduced into vectors suitable for large-scale protein production. A variety of these vectors are available from commercial suppliers, and any of them can be used.

[0200] For example, Figure 3 illustrates a plasmid map of ENPP1-2-1-exENPP3-Fc cloned into a pcDNA3 plasmid using an appropriate endonuclease restriction site. Protein subdomains are color-coded to illustrate the signal sequence of the fusion protein, the extracellular domain of ENPP3, and the Fc domain. The amino acid sequence of the cloned protein is also shown below the plasmid map, and this too is color-coded to illustrate the domains of the fusion protein. A pcDNA3 plasmid containing the desired protein construct can be introduced into an expression plasmid by stable transfection using established techniques such as electroporation or lipofectamine, and cells can be grown under antibiotic selectivity to increase the number of stably transfected cells.

[0201] Next, stably transfected single-cell clones are established and screened for high expression of the desired fusion protein. As previously described for ENPP1 (Saunders, et al., 2008, Mol. Cancer Therap. 7(10):3352-62; Albright, et al., 2015, Nat Commun. 6:10006), single-cell clones expressing ENPP3 protein can be screened in 96-well plates using a high-throughput method with the synthesis enzyme substrate pNP-TMP. Once high-expression clones are identified through screening, the protein can be produced in a shaking flask or bioreactor, as previously described for ENPP1 (Albright, et al., 2015, Nat Commun. 6:10006).

[0202] ENPP3 can be purified using a combination of standard purification methods known in the art. These techniques are well known in the art and can be selected from techniques such as column chromatography, ultracentrifugation, filtration, and precipitation. Column chromatography purification can be achieved using affinity chromatography, e.g., protein A and protein G resins, metal affinity resins, e.g., nickel or copper, hydrophobic exchange chromatography, and reversed-phase high-pressure chromatography (HPLC) using C8-C14 resins. Ion exchange chromatography, e.g., anion exchange chromatography and cation exchange chromatography, may also be used with commercially available resins, e.g., Q-sepharose (anion exchange) and SP-sepharose (cation exchange), blue sepharose resin and blue-sephadex resin, and hydroxyapatite resin. Size exclusion chromatography using commercially available S-75 and S200 Superdex resins can also be used, as is known in the art. The buffers used to solubilize the protein and supply the selective medium for the above chromatography steps are standard biological buffers known to those skilled in the art of protein chemistry.

[0203] Some examples of buffers used in the preparation include citrate buffer, phosphate buffer, acetate buffer, tris(hydroxymethyl)aminomethane buffer, saline buffer, glycine-HCl buffer, cacodylate buffer, and sodium barbital buffer, all of which are well known in the art. Using one technique or a combination of techniques and a suitable buffer system adjusted to the appropriate pH, the described fusion protein can be purified from the crude material to a purity of over 99% (see, for example, Figure 2). In this figure, partially purified ENPP3 and the crude starting material are compared side by side on a Coomassie-stained polyacrylamide gel after one purification step. As demonstrated in Figure 2, the crude starting material shown in the right lane after one purification step was enriched with protein with a molecular weight slightly exceeding 105 kD, corresponding to the appropriate molecular weight of ENPP3. This material can then be further purified using further techniques and / or chromatographic steps, as described above, to achieve a fairly high purity, such as about 99% purity. In certain embodiments, the purified protein exhibits enzymatic activity equivalent to that described and demonstrated in Figures 1A-1C.

[0204] Example 4: Use of plasma PPi as a biomarker Certain aspects of the present invention intend to use plasma pyrophosphate as a biomarker to determine which individuals are at risk of developing ectopic soft tissue calcification, vascular wall calcification, low bone density, osteopenia, stroke, arthritis, and / or hereditary rickets. Plasma pyrophosphate has never been used clinically to predict individuals at risk of the above disorders, as evidenced by the absence of plasma PPi testing in the clinical laboratory catalogs provided by leading clinical laboratories, e.g., Mayo Medical Laboratory (www.mayomedicallaboratories.com / test-catalog / alphabetical / P) or Yale University, or leading private reference laboratories, e.g., ARUP (ltd.aruplab.com / Search / Browse / P) or The Quest Diagnostics Nichols Institute (www.specialtylabs.com / about_us / ).

[0205] In certain aspects, plasma PPi has clinical utility as a predictor and diagnostic agent for identifying individuals at risk of calcification, ossification disorders, stroke, osteopenia, low bone density, and / or arthritis.

[0206] Plasma PPi can be measured using several published methods, including radioisotope methods (Cheung, et al., 1977, Anal. Biochem. 83(1):61-3) and fluorescence methods (Jansen, et al., 2013, PNAS USA 110(50):20206-11; Jansen, et al., 2014, Arterioscler. Thromb. Vasc. Biol. 34(9):1985-9). Accurate measurement of plasma PPi requires that platelets be removed from the plasma and that the whole blood is not hemolyzed when collected. Platelets can be removed from the blood by high-speed centrifugation or ultrafiltration. Platelet removal is necessary to prevent artificial increases in plasma PPi levels, as platelets release PPi and ATP into the plasma during activation and degranulation. Hemolysis of whole blood also releases ATP into the plasma, leading to erroneous increases in plasma PPi measurements. Plasma collected from un-hemolyzed blood and with platelets removed can be reliably used to measure PPi concentrations and can provide clinical utility as a predictive diagnostic agent to identify patients at risk of the aforementioned disorders.

[0207] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed in relation to a particular aspect, it will be apparent that other aspects and variations of the invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed as encompassing all such aspects and equivalent variations.

Claims

1. Isolated polypeptide of formula (I) or its pharmaceutically acceptable salt or solvate: Export-protein-Z-domain-XY(I) During the ceremony, The export function does not exist. The protein (PROTEIN) contains the extracellular domain (SEQ ID NO:1) of ENPP3. The domain is selected from the group consisting of human IgG Fc domains and human albumin domains. X is an existence hazard, Z is either absent or a linker consisting of 1 to 20 amino acids, and Y does not exist: A pharmaceutical composition for increasing plasma pyrophosphate (PPi) levels in individuals exhibiting plasma PPi levels lower than normal plasma pyrophosphate (PPi) levels, comprising: The isolated polypeptide of formula (I) or a pharmaceutically acceptable salt or solvate thereof has ATP hydrolase activity. The aforementioned pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the individual has at least one disease or disorder selected from the group consisting of systemic arteriosclerosis of infants (GACI), idiopathic arteriosclerosis of infants (IIAC), pseudoxanthoma elasticum (PXE), OPLL, hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic lesions, hereditary and non-hereditary osteoarthritis, ankylosing spondylitis, age-related arteriosclerosis, and calciphylaxis due to end-stage renal disease (ESRD) or bone mineral metabolism disorders associated with chronic kidney disease.

3. The pharmaceutical composition according to claim 1, wherein the protein (PROTEIN) nuclease domain is absent.

4. The pharmaceutical composition according to claim 1, wherein Z is selected from the group consisting of a linker consisting of 20 amino acids, a linker consisting of 19 amino acids, a linker consisting of 18 amino acids, a linker consisting of 17 amino acids, a linker consisting of 16 amino acids, a linker consisting of 15 amino acids, a linker consisting of 14 amino acids, a linker consisting of 13 amino acids, a linker consisting of 12 amino acids, a linker consisting of 11 amino acids, a linker consisting of 10 amino acids, a linker consisting of 9 amino acids, a linker consisting of 8 amino acids, a linker consisting of 7 amino acids, a linker consisting of 6 amino acids, a linker consisting of 5 amino acids, a linker consisting of 4 amino acids, a linker consisting of 3 amino acids, a linker consisting of 2 amino acids, and a linker consisting of 1 amino acid.

5. The pharmaceutical composition according to any one of claims 1 to 4, which is administered to the individual locally, locally, or systemically.

6. The pharmaceutical composition according to claim 1, wherein the domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, or the domain is a human albumin domain.

7. The pharmaceutical composition according to claim 6, wherein the domain is a human IgG Fc domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

8. The pharmaceutical composition according to claim 6, wherein the domain is a human albumin domain.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the individual is a mammal.

10. The pharmaceutical composition according to claim 9, wherein the mammal is a human.

11. The pharmaceutical composition according to claim 1, wherein the amount of the isolated polypeptide or a pharmaceutically acceptable salt or solvate thereof administered to the individual is an amount effective in raising the plasma PPi level in the individual to at least 800 nM.

12. The pharmaceutical composition according to claim 11, wherein the amount of the isolated polypeptide or a pharmaceutically acceptable salt or solvate thereof administered to the individual is an amount effective in raising the plasma PPi level in the individual to at least 1 μM or at least 1.5 μM.

13. The pharmaceutical composition according to any one of claims 11 to 12, wherein the individual has at least one disease selected from the group consisting of systemic arteriosclerosis of infants (GACI), idiopathic arteriosclerosis of infants (IIAC), pseudoxanthoma elasticum (PXE), ossification of the posterior longitudinal ligament (OPLL), vascular wall calcification (MWVC), autosomal recessive hypophosphatemic rickets type 2 (ARHR2), end-stage renal disease (ESRD), bone mineral metabolism disorder associated with chronic kidney disease (CKD-MBD), X-linked hypophosphatemia (XLH), age-related osteopenia, uremic arteriosclerosis of microarteriosclerosis (CUA), and hypophosphatemic rickets.

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